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Univers
ity of
Cap
e Tow
n
IDENTIFICATION AND PRELIMINARY CHARACTERIZATION OF THE 2,5-
DIPHENYLOXAZOLE BIOSYNTHETIC PATHWAY IN STREPTOMYCES POLYANTIBIOTICUS SPRT.
by
Ian Kyle Kemp
A THESIS PRESENTED FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY IN THE DEPARTMENT OF MOLECULAR AND CELL
BIOLOGY
FACULTY OF SCIENCE
UNIVERSITY OF CAPE TOWN
JULY 2015
The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non-commercial research purposes only.
Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author.
Univers
ity of
Cap
e Tow
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i
TABLE OF CONTENTS
Acknowledgements .................................................................... ii
Abstract ..................................................................................... iii
Abbreviations ............................................................................ vi
CHAPTER 1 General introduction .................................................................. 1
CHAPTER 2 Early attempts at isolating the gene cluster responsible for DPO
biosynthesis in Streptomyces polyantibioticus SPRT ............... 59
CHAPTER 3 Streptomyces polyantibioticus SPRT genome exploration...... 101
CHAPTER 4 Development of a transformation protocol for Streptomyces
polyantibioticus SPRT and gene disruption experiments ....... 151
CHAPTER 5 General discussion ................................................................. 217
APPENDIX A ............................................................................................... 247
APPENDIX B ............................................................................................... 248
APPENDIX C ............................................................................................... 256
ii
ACKNOWLEDGEMENTS
The following people and organisations are thanked:
My supervisor, Dr Paul Meyers, for his assistance and support throughout the
duration of this project.
All the members of Lab 202/3 for their continuous support and advice, with
special mention to Gareth Everest and Sarah Curtis. It has been a pleasure
working with all of you.
The financial assistance of the National Research Foundation (NRF) towards
this research is hereby acknowledged. Opinions expressed and conclusions
arrived at, are those of the author and are not necessarily to be attributed to the
NRF.
The Medical Research Council and the University of Cape Town for the
financial support that made this project possible.
Dr P. Whitney Swain III for providing the plasmid vector pJN100 and E. coli
ET12567/pUZ8002 that were used in this study.
Dr Bohdan Ostash for providing the plasmid vector pOJ260 that was used in
this study and for the advice on Streptomyces transformation.
My parents, Ian and Anne-Marie, for their love, financial support and guidance,
especially during tough times.
To all academic staff, scientific officers and departmental assistants for all their
help and assistance.
iii
IDENTIFICATION AND PRELIMINARY CHARACTERIZATION OF THE 2,5-
DIPHENYLOXAZOLE BIOSYNTHETIC PATHWAY IN STREPTOMYCES POLYANTIBIOTICUS SPRT.
by
Ian Kyle Kemp
Department of Molecular and Cell Biology, University of Cape Town, Private
Bag, Rondebosch, 7701, South Africa
ABSTRACT
An antibacterial compound produced by the actinomycete, Streptomyces
polyantibioticus SPRT, exhibited antibiosis against Mycobacterium tuberculosis
H37RvT (the causative agent of tuberculosis), which prompted interest in its
biosynthesis. The antibacterial compound was isolated in a previous study and its
structure was determined by X-ray crystallography and nuclear magnetic resonance
(NMR) to be 2,5-diphenyloxazole (DPO). Based on the structure of DPO, a biosynthetic
scheme for the synthesis of this molecule was proposed, whereby a non-ribosomal
peptide synthetase (NRPS) condenses a molecule of benzoic acid with 3-
hydroxyphenylalanine. The dipeptide is converted to a diphenyloxazole derivative by
heterocyclization and a final decarboxylation step leads to DPO. To determine whether
the hypothesis pertaining to the DPO biosynthetic pathway was correct, initial efforts
were made to identify the genes coding for benzoic acid synthesis and the DPO NRPS
in the S. polyantibioticus SPRT genome using PCR amplification, Southern
hybridization and sequencing. This led to the identification of 12 unique adenylation
(A) domains (of which one was specific for phenylalanine) and a gene, paaK, encoding
a phenylacetate CoA-ligase (PA-CoA), putatively involved in benzoic acid
biosynthesis. However, no further sequence information could be obtained for the
genes encoding the phenylalanine-specific A domain or PA-CoA and similar attempts
to identify other NRPS-associated domains, as well as genes involved in benzoic acid
synthesis, proved unsuccessful. In light of these difficulties, the S. polyantibioticus
iv
SPRT genome was sequenced and a gene cluster was identified as being responsible for
the biosynthesis of DPO using a genome mining approach. However, contrary to the
hypothesis that a linear NRPS system for DPO biosynthesis would be identified, the
gene cluster exhibited a nonlinear arrangement. The core domains are arranged as A-
PCP-C (instead of C-A-PCP) and there is also a stand-alone heterocyclization (Cy)
domain, a stand-alone thioesterase (TE) domain and an acyl-CoA synthetase putatively
involved in activating benzoic acid. Furthermore, there are two NRPS domains in the
gene cluster that are believed to be inactive. A possible biosynthetic pathway for
benzoyl-CoA production, encoded by a separate gene cluster, was identified based on
the genome analysis of S. polyantibioticus SPRT. In order to confirm the involvement
of the identified genes in DPO biosynthesis, an intergeneric conjugation protocol was
developed for the introduction of plasmid DNA into S. polyantibioticus SPRT and
subsequent gene disruption experiments. The putative DPO biosynthetic genes were
insertionally activated via homologous recombination and the method for isolating
DPO was carried out on each of the mutant strains, after which the extracts were assayed
for activity against Mycobacterium aurum A+ using TLC-bioautography analysis. The
absence of activity against M. aurum A+ in the extracts from mutant strains S.
polyantibioticus ∆A99, S. polyantibioticus ∆CYC and S. polyantibioticus ∆ACY
suggested the involvement of the A domain encoded by gene SPR_53060, the putative
Cy domain encoded by gene SPR_53040 and the acyl-CoA synthetase encoded by gene
SPR_52860 in the biosynthesis of DPO. However, attempts to identify the genes
responsible for benzoic acid biosynthesis proved unsuccessful, as gene disruption did
not abolish DPO activity in the S. polyantibioticus ∆LAC, S. polyantibioticus ∆PAAK
and S. polyantibioticus ∆CIN mutant strains encoding the putative D-lactate
dehydrogenase encoded by gene SPR_60250, the PA-CoA ligase (paaK) encoded by
gene SPR_46390 and the cinnamate-CoA ligase encoded by gene SPR_60150,
respectively. Based on the genome annotation analysis and gene disruption studies, a
model for DPO biosynthesis is proposed. At this stage, the model cannot account for
the source of benzoic acid, as in vivo gene disruption experiments disproved both of the
hypotheses on how benzoic acid is synthesized in S. polyantibioticus SPRT. However,
alternative hypotheses regarding the mechanism of benzoic acid biosynthesis in S.
polyantibioticus SPRT are proposed and are suggested as the place to start in future
studies to elucidate the production of this unusual starter unit in DPO biosynthesis.
Furthermore, the identification of the gene cluster responsible for DPO biosynthesis has
v
laid the foundation for future combinatorial biosynthetic studies to create derivatives of
DPO that might be used in the treatment of drug resistant tuberculosis. Lastly, the S.
polyantibioticus SPRT genome sequence could be explored for the identification of
antibiotic gene clusters for other potential antitubercular antibiotics that this organism
produces.
vi
ABBREVIATIONS
α alpha β beta ∆ delta γ gamma λ lambda Ω ohm (s) Φ phi µg microgram(s) µl microlitre(s) µF microFarad (s) µM micromolar °C degrees Celsius % percentage A adenylation domain aprR apramycin resistant AIDS Acquired Immunodeficiency Syndrome ACP acyl carrier protein ArCP aryl carrier protein ATP adenosine triphosphate AMP adenosine monophosphate AMT aminotransferase BC Before Christ bp base pair(s) BLAST Basic Local Alignment Search Tool C condensation domain Cy heterocyclization domain CDC Centers for Disease Control and Prevention CDD Conserved Domain Database clt cis-acting locus of transfer COGs clusters of orthologous groups COM communication-mediating CoA Coenzyme A DMSO dimethyl sulphoxide DNA deoxyribonucleic acid dsDNA double-stranded DNA ssDNA single-stranded DNA
vii
dH20 distilled water DPO 2,5-diphenyloxazole dNTP deoxyribonucleoside triphosphates (dATP, dCTP, dTTP and dGTP) et al. and others (et alii) E epimerization domain FAS fatty acid synthase FMN flavin mononucleotide g gram(s) gDNA genomic DNA GrsA gramicidin synthetase HAL histidine ammonia-lyase HIV Human immunodeficiency virus HM Hacène’s medium HMM Hidden Markov Model HPLC High-performance liquid chromatography h hour(s) Inc. Incorporated IncP Incompatibility group P ISP International Streptomyces Project IPTG isopropyl-β-D-thiogalactopyranoside kb kilobase(s) kg kilogram(s) kDa kiloDalton(s) kV kiloVolt(s) l litre LA Luria-Bertani agar LB Luria-Bertani broth M molar MDR multidrug-resistant MEGA Molecular Evolutionary Genetics Analysis ML maximum likelihood MP maximum parsimony MRSA methicillin-resistant Staphylococcus aureus MS mannitol soya MT methyltransferase Mb mega base(s)
viii
mg milligram(s) min minute(s) ml millilitre(s) mM millimolar mm millimetre(s) mRNA messenger RNA ms millisecond (s) mA milliAmp(s) NADP Nicotinamide adenine dinucleotide phosphate NGS Next-generation sequencing NJ neighbour joining NRPS Non-ribosomal peptide synthetase NRP Non-ribosomally synthesized peptide ng nanogram(s) nm nanometre(s) OD optical density ORF open reading frame oriT origin of transfer Ox oxidation domain O/N overnight PA phenylacetate PA-CoA phenylacetate-CoA ligase PAL phenylalanine ammonia-lyase PCP peptidyl carrier protein PCR polymerase chain reaction PDR pan drug-resistant PEG polyethylene glycol PKS Polyketide Synthase PPi inorganic pyrophosphate PPTase phosphopantetheinyltransferase PP phosphopantetheine p plasmid RAST Rapid Annotation using Subsystem Technology RE reduction domain Rf retention factor REase restriction endonuclease RSA Republic of South Africa R-M restriction-modification RNA ribonucleic acid RNase ribonuclease
ix
rpm revolutions per minute s second(s) SEM standard error of the mean SAM S-adenosylmethionine SDS sodium dodecyl sulphate SSC standard saline citrate spp. species (plural) T type strain TB Tuberculosis TE thioesterase domain TLC thin layer chromatography TSB tryptic soy broth TSVM transductive support vector machine U unit(s) USA United States of America UK United Kingdom UV ultraviolet VRSA vancomycin-resistant Staphylococcus aureus v/v volume per volume V volt(s) WHO World Health Organisation XDR extensively drug-resistant YEME yeast extract-malt extract medium (ISP Medium No.2) ZMW zero-mode waveguide
2
CHAPTER 1
GENERAL INTRODUCTION
1.1 Antibiotic resistance........................................................................................... 3
1.2 The phylum Actinobacteria ............................................................................... 9
1.3 Natural product discovery ................................................................................ 11
1.3.1 Oxazole-containing natural prdoucts ................................................... 14
1.4 Non-ribosomal peptide synthetases (NRPSs) .................................................. 17
1.4.1 Assembly logic of NRP synthesis ........................................................ 20
1.4.1.1 Activation by the Adenylation domain .................................... 21
1.4.1.2 Transport of substrates and intermediates to the catalytic
centres by the Peptidyl Carrier Protein .................................... 23
1.4.1.3 Peptide elongation by the Condensation domain ..................... 24
1.4.1.3.1 Heterocyclization domains............................... 27
1.4.1.4 Peptide release by the Thioesterase domain ............................ 29
1.4.1.5 Editing/tailoring domains......................................................... 31
1.4.1.5.1 Methylation ...................................................... 31
1.4.1.5.2 Epimerization (control of stereochemistry) ..... 32
1.4.1.5.3 Reduction domain ............................................ 32
1.4.1.5.4 Oxidation domain............................................. 33
1.4.1.5.5 Further modifications ....................................... 34
1.4.2 NRPS substrate specificity prediction and the non-ribosomal code .... 35
1.4.3 NRPS biosynthetic strategies ............................................................... 41
1.4.4 Combinatorial biosynthesis and intermolecular communication ......... 45
1.5 Research aims .................................................................................................. 47
1.6 Reference list ................................................................................................... 50
Chapter 1 - Introduction
3
CHAPTER 1
GENERAL INTRODUCTION
The existence of antimicrobial agents was first described in 1877 when Louis Pasteur
and Robert Koch observed that one type of bacterium could prevent the growth of
another. The term “antibiosis’ was introduced by the French bacteriologist, Vuillemin,
as a descriptive name for this phenomenon. In 1942, Selman Waksman, an American
microbiologist, devised the term antibiotic to describe any substance produced by a
microorganism that is antagonistic to the growth of other microorganisms in high
dilution (Waksman, 1947). The value of antibiotics to medicine was soon realized and
it lead to the heightened interest in the isolation and discovery of these novel
compounds. Indeed, the rapid development of antimicrobial agents over the past
century has greatly improved the treatment of infection and disease. However, the
increased usage of these drugs in the 1950s sparked the arms race between humanity
and microbial pathogens, as increased examples of recalcitrant infections due to the
inevitable rise of drug-resistant pathogens became apparent (Davies, 2012; Davies,
2007).
1.1 ANTIBIOTIC RESISTANCE
The use of antibiotics as a means of treating infectious disease in humans and food-
producing animals has revolutionized medicine and become widespread since the
introduction of penicillin in the 1940s. Regrettably, the extensive misuse of these drugs
in both the clinical and agricultural setting has led to the rapid emergence of drug-
resistant strains of bacteria and other microbes. This resistance has led to a decrease in
the effectiveness of currently available antimicrobial drugs and the problem is further
compounded by the emergence of multidrug-resistant microbial strains.
Chapter 1 - Introduction
4
Microorganisms that have developed resistance to several different antibiotics are
referred to as multidrug-resistant (MDR) strains.
It was reported in 2004 that more than 70 % of pathogenic bacteria were predicted to
be resistant to at least one of the currently available antibiotics. Although we have
witnessed a steady increase in resistance in almost every pathogen to most of the
currently available antibiotics, not all of these antibacterial agents display the same rate
of resistance development. Indeed, single target drugs such as rifampicin are more
susceptible to the development of resistance than drugs that inactivate several targets
irreversibly, such as penicillin (Demain & Sanchez, 2009; Spratt, 1994).
Global healthcare systems are also encountering extensively drug-resistant (XDR)
organisms on a regular basis and these microorganisms are resistant to all antibiotics
except colistin, a highly toxic agent, which was abandoned in the 1960s due to its
questionable efficacy. In the most severe case, certain microorganisms are pan drug-
resistant (PDR), meaning that they are resistant to all available antibiotics (Udwadia &
Vendoti, 2013; Udwadia, 2012).
It has been reported that at least 2 million people become infected with antibiotic-
resistant bacteria each year in the United States, with at least 23 000 people dying as a
direct result of these infections. It is also believed that most deaths related to antibiotic
resistance happen in healthcare facilities such as nursing homes and hospitals. One of
the most common causes of healthcare-associated infections is methicillin-resistant
Staphylococcus aureus (MRSA), claiming the lives of more than 11 000 people in the
United States in 2011. This MDR, Gram-positive pathogen is resistant to all penicillins
and cephalosporins (i.e. all β-lactam antibiotics), as well as often being resistant to
clindamycin and quinolones. S. aureus bacteria produce a biofilm which protects them
from the environment. These biofilms can grow on wounds, scar tissue and medical
implants or devices (Dantas & Sommer, 2014). It is estimated that more than 70 % of
the bacterial species that produce biofilms are likely to be resistant to at least one of the
drugs commonly used in anti-infectious therapy in hospitals (Demain & Sanchez,
2009). Once described as only a hospital-inhabiting or nosocomial infection, the rate
of MRSA infections has increased rapidly among the general population in the past
Chapter 1 - Introduction
5
decade, which further exacerbates the urgent need for new therapeutic agents (Centers
for Disease Control and Prevention, 2013).
Vancomycin has long been the treatment of choice for MRSA infections, however, new
treatments were sought after the Centers for Disease Control and Prevention (CDC)
reported the first case of S. aureus resistant to both methicillin and vancomycin in 2002.
Since then, the number of reported cases of vancomycin-resistant S. aureus (VRSA)
has remained relatively low and thus the epidemiology and risk factors associated with
VRSA are not completely understood. Further research is required in order to
understand these aspects, as well as the implications pertaining to clinical and infection
control and optimal treatment (Howden et al., 2010; Applebaum, 2006).
Pathogens that are less prevalent than MRSA, but that pose a threat of infection that is
genuinely untreatable are those such as the carbapenem-resistant Enterobacteriaceae.
Within this large family of Gram-negative bacteria, strains of Acinetobacter baumannii,
Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa are currently
tormenting global healthcare systems due to their resistance to all β-lactam antibiotics.
This resistance is conferred by the ability of their outer membrane to prevent the entry
of the antibiotics, in addition to being able to expel, via efflux pumps, the remainder
that does successfully enter the cell (Fischbach & Walsh, 2009).
Furthermore, a report released by the CDC in 2013 identified the pathogens Clostridium
difficile and Neisseria gonorrhoeae as antibiotic-resistant bacteria that require urgent
public health attention in order to identify infections and limit transmission (CDC,
2013). The report also highlighted the serious threat of MDR and XDR strains of
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB).
Tuberculosis is a contagious airborne disease and is second only to human
immunodeficiency virus (HIV)/AIDS as the leading killer worldwide due to a single
infectious agent (Liu et al., 2012). Approximately 9 million people contracted TB in
2013, of which 1.5 million people died from the disease. About 480 000 people
developed MDR-TB globally in 2013, an increase of almost 7 % from 2012, of which
more than half the cases were in developing countries such as India, China and the
Chapter 1 - Introduction
6
Russian Federation. It was also estimated that 9 % of the MDR-TB cases had XDR-
TB (World Health Organisation, 2014)
In the majority of cases, TB is treatable and curable with the available first-line anti-
TB drugs (rifampicin, isoniazid and pyrazinamide). However, disease caused by M.
tuberculosis strains that are resistant to one or more of these standard drugs is far more
challenging to treat, largely due to the higher cost, adverse side-effects and longer
treatment regimes of the available second-line drugs. MDR-TB strains are defined as
being resistant to at least two of the first-line anti-tubercular drugs, isoniazid and
rifampicin. XDR-TB strains are defined as being resistant to both these drugs as well
as to any fluoroquinolone and at least one of the injectable second-line drugs (i.e.
kanamycin, amikacin or capreomycin). Extensively drug-resistant TB has been
identified in 92 countries and in all regions of the world (WHO, 2014; CDC, 2013;
Rivers & Mancera, 2008; Petrini & Hoffner, 1999).
The primary cause of MDR-TB and XDR-TB is incomplete or incorrect treatment,
which is mainly attributed to patients not completing their full course of antibiotic
therapy, but is also due to the usage of inappropriate drug combinations, poor treatment
compliance, using single drugs for ordinary TB, clinics running out of drug stocks and
the use of poor quality medicines (WHO, 2014; Cape Gateway, 2006).
The World Health Organisation (WHO) has predicted that between 2000 and 2020,
almost 1 billion people globally will contract tuberculosis and that the disease will claim
the lives of approximately 35 million people. Twenty-two high-burden countries were
identified, which account for 81 % of all estimated TB cases that occur globally. South
Africa has the highest incidence of TB in the world per capita population and this
problem is compounded by the high incidence of HIV co-infection, as HIV-positive
individuals infected with M. tuberculosis have a dramatically increased chance of
developing TB compared to TB-infected, HIV-negative individuals (Churchyard et al.,
2014; Bloom and Murray, 1992). South Africa also has the largest number of HIV-
associated TB cases and the second-largest number of MDR-TB cases, after India.
Notable progress has been achieved in reducing TB prevalence and deaths and
improving treatment outcomes for new TB cases in recent years, but the burden still
remains enormous, especially considering the drastic rise in the numbers of MDR and
Chapter 1 - Introduction
7
XDR strains. New TB drugs and vaccines are urgently required to further accelerate
progress towards improved TB control in South Africa and other countries (Churchyard
et al., 2014).
Consequently, there is a global healthcare emergency, as new antibiotic discovery and
subsequent treatment options have been outpaced by the emergence of drug-resistant
microorganisms. In order to control the global TB epidemic, there is an urgent need
for new and improved TB drugs. These new drugs should, ideally, be effective against
both MDR and XDR M. tuberculosis strains, be able to shorten the duration of treatment
from the current six month regime and simplify treatment by reducing the number of
pills that need to be taken each day, as well as reducing the dosage frequency to once a
week. In addition, new TB drugs should be able to be administered simultaneously
with HIV drugs and be effective against latent TB (Koul et al., 2011; Lamichhane,
2011; Barry et al., 2009; Young et al., 2009; Rivers and Mancera, 2008).
In light of this, taxonomy-guided bacterial bioprospecting needs to continue to play an
important role in identifying new microbial natural products, such as bacterial
secondary metabolites that have already served the pharmaceutical industry well as TB
drugs. It has been estimated that less than 10 % of the world’s biodiversity has been
tested for biological activity, so there is a high likelihood of isolating new antibiotic
molecules from bacteria (Ashforth et al., 2010; Harvey, 2000).
In addition, the relentless evolution of antibiotic-resistant strains of microbial
pathogens erodes the utility of the classical antibiotics and necessitates the perpetual
need for discovery of new antibiotics (Ashforth et al., 2010). Consequently, numerous
research groups are now focussing on identifying novel antibiotics in new species of
Streptomyces and other genera of Actinobacteria due to the fact that they are renowned
for their ability to produce antibiotics and other bioactive natural products with a wide
range of applications in medicine and agriculture. Examples of these compounds
include antibacterials such as erythromycin A, vancomycin and daptomycin,
antifungals such as amphotericin B, immunosuppressants such as FK-506, anticancer
agents such as doxorubicin and epoxomicin, anthelmintics such as avermectin,
insecticides such as spinosyn A and herbicides such as phosphinothricin (Figure 1.1)
(Challis, 2014).
Chapter 1 - Introduction
8
Figure 1.1 Examples of secondary metabolites used in medicine and agriculture produced by
Actinobacteria (Challis, 2014).
Chapter 1 - Introduction
9
1.2 THE PHYLUM ACTINOBACTERIA
The phylum Actinobacteria represents one of the largest taxonomic units within the
domain Bacteria and is predicted to be the most abundant source of small molecule
diversity on earth (Miao & Davies, 2010). The phylum consists of Gram-positive
bacteria with typically elevated guanosine (G) and cytosine (C) content in their
chromosomal DNA, ranging from 50 % in certain corynebacteria to more than 70 % in
Streptomyces and Frankia species (Ventura et al., 2007).
Actinobacteria are inhabitants of soil, the rhizosphere, marine and extreme arid
environments and exhibit a wide range of morphologies, including coccoid, rod-shaped
and hyphal forms. The filamentous actinobacteria are often referred to as
actinomycetes and produce branching hyphae which form a mycelium. In many genera
of actinomycetes, some of the vegetative hyphae differentiate into arthrospores. Some
genera of actinomycetes exhibit fragmentation in liquid and/or plate culture (e.g.
Amycolatopsis), a phenomenon in which the vegetative hyphae break down into rod-
shaped or coccoid elements. Additionally, actinobacteria are able to produce a variety
of extracellular enzymes and secondary metabolites as a result of the regulated
expression of gene clusters (MacNeil et al., 1992; Chater, 1990). Interest in the phylum
intensified after the discovery of streptomycin in the laboratory of Selman Waksman in
1943 and the subsequent observation that many of the secondary metabolites are indeed
potent antibiotics. In particular, Streptomyces species have been exploited extensively
by the pharmaceutical industry as a primary source of natural products for use as
therapeutic agents, but bacteria belonging to the suborders Micromonosporineae (e.g.
Micromonospora), Pseudonocardineae (e.g. Amycolatopsis) and Streptosporangineae
(e.g. Planobispora) have also been described as abundant producers of novel
antibacterial antibiotics (Ashforth et al., 2010; Miao & Davies, 2010; Ventura et al.,
2007).
Actinobacterial genomes, particularly those of the actinomycetes (order
Actinomycetales), are larger than most other bacteria, with sizes ranging from 0.93 Mb
(Tropheryma whipplei) to 11.9 Mb (‘Streptomyces bingchenggensis’) and therefore
possess a high capacity for secondary metabolite production (Verma et al., 2013).
Chapter 1 - Introduction
10
Among the actinomycetes, such as Streptomyces, it is estimated that more than 60 % of
the secondary metabolites produced are synthesized by enzymatic systems known as
non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) or mixed
NRPS-PKS pathways (Baltz, 2014). Moreover, it is widely accepted that species that
possess a large number of NRPS and/or PKS genes produce more secondary
metabolites. For example, the sequencing of the first antibiotic-producing
actinomycete genomes, those of Streptomyces coelicolor strain A3(2) (8.6 Mb) and
Streptomyces avermitilis strain MA-4680T (9.02 Mb), revealed the potential of both
organisms to produce a large number of secondary metabolites, most of which were not
synthesized under standard growth conditions (Baltz, 2008).
The genome sequence of S. avermitilis MA-4680T revealed 24 PKS and NRPS clusters
for previously unidentified secondary metabolites and in the case of S. coelicolor A3(2),
a genome mining approach identified the capability of the organism to produce twice
as many secondary metabolites as was originally thought (Nett et al., 2009; Bentley et
al., 2002). Similarly, annotation of the genomes of S. avermitilis MA-4680T and
Streptomyces griseus strain NBRC 13350, revealed that 60 % and 75 %, respectively,
of the biosynthetic gene clusters in these organisms encoded unidentified secondary
metabolites, further suggesting that natural product biosynthetic capacity has been
immensely underestimated (Challis, 2014; Nett et al., 2009; Walsh, 2004; Challis &
Ravel, 2000).
The sequenced actinobacterial genomes (currently numbering 4059) (GenBank, 2015;
Land et al., 2015) are an invaluable resource which has revealed that the more bacterial
genomes that are sequenced, the more new gene families are discovered, thus
uncovering new genetic diversity and thereby new biosynthetic capabilities (Wu et al.,
2009). This genetic diversity is directly afforded by NRPS multifunctional enzymatic
systems that are able to manufacture structurally diverse natural products with a
remarkable variety of useful/significant pharmacological activities (Ashforth et al.,
2010; Wu et al., 2009).
Chapter 1 - Introduction
11
1.3 NATURAL PRODUCT DISCOVERY
Natural products, which can be produced from primary or secondary metabolism by
living organisms such as bacteria, plants, mammals, marine invertebrates, insects and
fungi, are defined as low molecular weight, organic molecules (Davies & Ryan, 2012).
The bacterial metagenome contributes to the production of primary metabolites and
conversion of small molecules into secondary metabolites, also called “specialized
metabolites”. Two of the important classes of secondary metabolites classified as
natural products are the polyketides and non-ribosomal peptides. Other structural
classes include alkaloids, terpenoids, aminoglycosides and shikimate-derived
molecules (Baltz, 2014; Davies & Ryan, 2012).
For over half a century, natural products have played an important role as targets of
study for analytical and synthetic chemists, as chemical tools for probing biological
systems to aid in discovering the roles of individual biomolecules, but most
importantly, they have acted as a treasure trove of compounds used to treat infectious
diseases in humans, animals and crops (Davies, 2011; Fischbach & Walsh, 2009).
Indeed, it has been estimated that approximately 40 % of all medicines in clinical use
are either natural products or their semi-synthetic derivatives (John, 2009). This may
not come as a surprise, considering that herbal medicine formed the cornerstone of
sophisticated traditional medicine practices, with the earliest records dating back to
2600 BC in Mesopotamia, where plant-derived substances such as the oils of Cupressus
semevirens (cypress), Cedrus species (cedar) and Glycyrrhiza glabra (licorice) where
used to treat ailments such as coughing, parasitic infections and inflammation (Cragg
& Newman, 2013; John, 2009). It has also been documented that the Greeks and
Romans contributed significantly to the development of the use of herbal medicine in
the ancient Western world (Cragg & Newman, 2013).
Furthermore, the serendipitous discovery of morphine in 1805 was the first
pharmacologically active pure compound isolated from a plant. This organic alkaloid
was derived from the resinous gum secreted by Papaver somniferum (the opium poppy)
and sparked the study of alkaloid chemistry, which inadvertently accelerated the
emergence of the modern pharmaceutical industry. Similar alkaloids from organic
Chapter 1 - Introduction
12
substances were isolated soon after, such as strychnine in 1817, caffeine in 1820 and
nicotine in 1828 (Li & Vederas, 2009).
In contrast, the microbial-derived drug era began in the 1920s with the discovery of the
antibiotic, penicillin, by Sir Alexander Fleming. Penicillin was later produced
industrially as a powder and used as a potent antibacterial compound during World War
II. The observation of its broad therapeutic use started what became known as “The
Golden Age of Antibiotics” and prompted the massive screening of microorganisms for
new antibiotics and related novel bioactive molecules (Cragg & Newman, 2013).
By 1990, 80 % of therapeutic medicines were natural products or analogues inspired by
them (Li & Vederas, 2009). Microorganisms remain a prolific source of structurally
and chemically diverse bioactive metabolites and these include antibacterial agents,
such as the penicillins (from Penicillium species), cephalosporins (from
Cephalosporium acremonium), aminoglycosides, tetracyclines and other polyketides of
many structural types (from members of the order Actinomycetales);
immunosuppressive agents, such as the cyclosporins (from Trichoderma and
Tolypocladium species) and rapamycin (from Streptomyces species); cholesterol-
lowering agents, such as mevastatin (compactin; from Penicillium species) and
lovastatin (from Aspergillus species); and anthelmintics and antiparasitic drugs, such
as the ivermectins (from Streptomyces species) (Cragg & Newman, 2013; Demain &
Sanchez, 2009; Li & Vederas, 2009).
Historically, the classical screening and bioassay-guided isolation techniques were very
successful in providing a route to the discovery of novel natural products for the
development of new therapeutic agents (Subramani & Aalbersberg, 2013). Over the
past two decades, however, natural product discovery efforts, particularly microbially-
produced products, have waned considerably due to the high rediscovery rate of known
compounds. Most of the common core structures from which today’s antibiotics are
derived were introduced between the mid-1930s and the early 1960s (Figure 1.2)
(Fischbach & Walsh, 2009). Consequently, pharmaceutical firms have preferred to
pursue alternative options in their search for new therapeutic agents, such as chemically
tailored derivatives of the classical scaffolds and modern techniques such as in silico
Chapter 1 - Introduction
13
screening, combinatorial biosynthesis (Cane et al., 1998) and combinatorial
biocatalysis (Watve et al., 2001; Michels et al., 1998).
Figure 1.2 No new major classes of antibiotics were introduced between 1962 and 2000 (referred
to as an innovation gap) (Fischbach & Walsh, 2009).
Despite this lack of interest, natural products continue to provide greater structural
diversity than standard combinatorial chemistry and therefore offer far more
opportunities for finding novel chemical scaffolds that are active against a wide range
of targets. In addition, the majority of the world’s biodiversity remains unexplored and
it has become increasingly apparent that approximately only 1 % of the microbial
diversity has been cultured and studied experimentally (Van Lanen & Shen, 2006;
Harvey, 2000). Attempts at exploring the uncultured or allegedly unculturable bacteria
using culture-independent methods such as metagenomics have been investigated and
it has become evident that these bacteria may in fact be culturable, thus providing a
potentially large source of new bioactive compounds (Watve et al., 2001; Handelsman
et al., 1998; Hugenholtz et al., 1998; Seow et al., 1997).
Chapter 1 - Introduction
14
Nevertheless, it was reported in 2005 that the number of discovered natural products
exceeds 1 million and this is mainly due to improvements in screening methods, as well
as separation and isolation techniques. Among these compounds, it is estimated that
50-60 % are produced by plants and 5 % are produced by microbes. Furthermore, it is
approximated that from the pool of biologically active compounds obtained from
microbes, 45 % are produced by actinomycetes, 38 % by fungi and 17 % by unicellular
bacteria (Bérdy, 2005).
There is still a gigantic proportion of undiscovered metabolites produced by the
actinomycetes and predictive modelling by Watve et al. (2001) suggested that more
than 150 000 bioactive metabolites are still waiting to be discovered from the members
of the genus Streptomyces alone. In order to gain access to the biodiversity afforded in
these and other actinobacterial suborders, it is imperative to continue efforts to isolate
novel actinobacteria from underexplored, diverse natural habitats.
There has been an increasing number of novel secondary metabolites isolated from
actinobacteria, specifically from the genus Streptomyces, over the past few decades,
and one such class is the heterocyclic-ring containing metabolites such as the oxazoles
and thiazoles. Several of these metabolites have been shown to exhibit extremely useful
biological properties, such as antimycobacterial activity (Walsh & Fischbach, 2010).
1.3.1 Oxazole-containing natural products
Oxazoles are aromatic 5-membered heterocycles which contain both nitrogen and
oxygen (Yeh, 2004). They are produced in nature by dramatic chemical modifications
performed on elongating peptide chains, most often catalysed by NRPS assembly lines
(Walsh et al., 2001). Oxazole biosynthesis is achieved via the cyclodehydration of
serine or threonine to yield a dihydroheteroaromatic oxazoline, which is then subjected
to a two-electron oxidation step and in turn yields the oxazole core structure (Figure
1.3). Reduction of the carbon-nitrogen bond in oxazoline creates an oxazolidine ring
(Yeh, 2004; Roy et al., 1999, Milne et al., 1998). All three of these oxidation states
can be found in natural products and if the starting residue is cysteine, the corresponding
process would yield thiazolines, thiazoles and thiazolidines (Roy et al., 1999; Milne et
al., 1998).
Chapter 1 - Introduction
15
Figure 1.3 Schematic representation of the biosynthetic process involved in oxazole and thiazole
production (Roy et al., 1999).
It was initially considered that naturally occurring oxazoles were rare, until heightened
research in the 1980s proved their ubiquity in nature (Yeh, 2004). The anticonvulsant
alkaloid, pimprinine, was one of the earliest oxazole-containing compounds to be
discovered, which was isolated from ‘Streptomyces pimprina’ in 1960 (Bhate et al.,
1960). Two related alkaloids, pimprinethine and pimprinaphine, were isolated almost
twenty years later from Streptomyces cinnamoneus and Streptomyces olivoreticuli,
respectively (Yoshioka et al., 1981). Furthermore, in 1966, the macrocyclic
streptogramin antibiotic accommodating a 2,4-disubstituted oxazole ring,
virginiamycin M2, was isolated from Streptomyces virginiae (Kondo et al., 1989). The
2,4-disubstituted oxazole ring is a common feature in several cyclic peptide natural
Chapter 1 - Introduction
16
products including the antileukemic metabolites, orbiculamide and keramide E, which
were both isolated from the marine sponge, Theonella sp., in 1991 (Fusetani et al.,
1991) and 1995 (Kobayashi et al., 1995), respectively. Additionally, in 1998, Rastogi
and colleagues reported the antimycobacterial activity of the oxazole alkaloid, texaline,
isolated from the plants Amyris texana and Amyris elemifera (Rastogi et al., 1998).
The stimulated interest in oxazoles (and thiazoles) can be attributed to their diverse
biological properties and their ability to interact with a wide variety of intracellular
bacterial targets, specifically proteins, DNA and RNA (Walsh, 2004; Milne et al.,
1998). Additionally, the oxazoline and thiazoline rings are important elements of
bioactive natural products and well known antimicrobial products. The use of these
rings as building blocks in pharmaceutical drug discovery is continually increasing and
one example is the phenyloxazolines which are able to inhibit the growth of certain
Gram-negative bacteria by impeding the production of lipid A, a molecule located in
the outer membrane of most Gram-negative bacteria (Padmavathi et al., 2009; Jackman
et al., 2000).
The biosynthesis of several oxazoles, thiazoles and their derivatives has been reported
to be catalysed by NRPS and NRPS/PKS hybrid enzyme complexes. Examples include
tallysomycin produced by Streptoalloteichus hindustanus ATCC 31158 (Tao et al.,
2007), virginiamycin produced by S. virginiae (Pulsawat et al., 2007) and zorbamycin
produced by Streptomyces flavoviridis ATCC 21892 (Wang et al., 2007). More
recently, novel antitrypanosomal antibiotics, the spoxazomicins, containing an oxazole
moiety within their structure, were isolated from the endophytic actinomycete,
Streptosporangium oxazolinicum. However, the biosynthetic machinery involved in
the synthesis of the spoxazomicins is yet to be elucidated (Inahashi et al., 2011).
Chapter 1 - Introduction
17
1.4 NON-RIBOSOMAL PEPTIDE SYNTHETASES (NRPSs)
Non-ribosomally synthesized peptides (NRPs) represent a class of microbial natural products
that exhibit unparalleled diversity in both structure and biological activity. These
characteristics can be attributed to the incorporation of many unusual, nonproteinogenic
residues and modifications (Konz & Marahiel, 1999; Marahiel, 1997). As the name implies,
NRPs are normally synthesized from amino acids, but over 400 monomers are known to be
used as substrates, which include non-proteinogenic amino acids such as ornithine and
hydroxyphenylglycine, D-configured and N-methylated amino acids, as well as a variety of
hydroxyl-amino acids, which can be further modified by acylation, glycosylation and
heterocyclic ring formation mediated by associated tailoring enzymes (Bloudoff et al., 2013;
Konz & Marahiel, 1999). Despite the structural diversity, however, the majority of NRPs share
a common biosynthetic origin, which has been linked to an enzymatic system known as the
“thiotemplate multienzymic mechanism” (Marahiel, 1997; Kleinkauf & von Döhren, 1990).
This enzymatic system is more commonly referred to as a non-ribosomal peptide synthetase
(NRPS) and these large, multifunctional enzymes range in size from 100-2000 kDa and can
catalyze up to several dozen reactions in a co-ordinated, assembly-line manner (Mootz et al.,
2002; Cane & Walsh, 1999; Marahiel, 1997).
Non-ribosomal peptide biosynthesis is widespread in a variety of bacterial and fungal species
and plays a significant role in the production of several biologically relevant products
(Schwarzer et al., 2003). These include antibiotics such as bacitracin A, surfactin,
tyrocidine A, gramicidin S and the penicillin precursor, isopenicillin N; immunosuppressive
agents such as cyclosporin (used in the aftercare of organ transplant patients); bleomycin A2
and epothilone (used in cancer therapy due to their cytostatic activity); and siderophores such
as enterobactin and myxochelin A (Figure 1.4) (Schwarzer et al., 2003: Marahiel, 1997).
Additionally, the genes encoding bacterial NRPSs are organised into operons, which are
usually 6-45 kilobases in length and encode protein templates that are often much smaller than
their fungal counterparts (Marahiel, 1997).
Chapter 1 - Introduction
18
Figure 1.4 The chemical structures of some bacterial (gramicidin S, bacitracin A, tyrocidine A, surfactin
and enterobactin) and fungal (cyclosporin A and isopenicillin) bioactive compounds whose
peptide backbones are synthesized by the non-ribosomal thiotemplate mechanism (adapted from
Marahiel, 1997).
More importantly, NRPSs are organised into repeated functional units known as modules, each
of which is responsible for one stage of polypeptide chain elongation leading to the formation
of the final non-ribosomal peptide product (Galm et al., 2008; Cane & Walsh, 1999; Marahiel,
1997). The number of modules within an NRPS generally corresponds to the number of amino
acids present in the structure of the peptide being synthesized and the modules are aligned in
such a way as to be co-linear with the corresponding peptide product (Lautru & Challis, 2004;
Marahiel, 1997). Additionally, each module consists of several structurally independent
domains, with each one catalysing a single reaction step such as activation, substrate
recognition, covalent bonding, optional modification of the incorporated amino acid monomer
and condensation with the amino acid acyl or peptidyl group on the neighbouring module
(Figure 1.5) (Mootz et al., 2002).
Chapter 1 - Introduction
19
Figure 1.5 Example of a NRPS assembly line consisting of seven modules responsible for the incorporation
of seven amino acids. Twenty-four chemical reactions are catalysed in the formation of the
final peptide product by twenty-four domains of five different types (C, A, PCP, E and TE)
(Sieber & Marahiel, 2005).
A basic elongation module consists of an adenylation (A) domain, a condensation (C) domain
and a peptidyl carrier protein (PCP) domain. The core of each module is the A domain that is
responsible for the activation of the cognate amino acid as an aminoacyl adenylate through
ATP hydrolysis. The activated substrate is then transferred onto the thiol moiety of the PCP
domain, which subsequently transports the substrate to the C domain. The C domain catalyzes
peptide bond formation between this amino acid substrate and the peptide attached to the PCP
domain of the preceding module, thus elongating the peptide chain. Following peptide bond
formation, the elongated peptide chain is attached to the PCP domain of the downstream
module, where it is passed off and further elongated in the next peptidyltransferase reaction.
The thioesterase (TE) domain, in the termination module, catalyzes the release (and in some
cases oligomerization and/or cyclization) of the mature NRPS-bound peptide product (Figure
1.6) (Bloudoff et al., 2013).
Chapter 1 - Introduction
20
Figure 1.6 A simplified example of a typical NRPS assembly line. (1) The cognate amino acids are
activated as aminoacyl-adenylates by the A domains. (2) The aminoacyl-adenylates are
transferred onto the PCP domains. (3) Sequential condensation of the PCP-bound amino acids.
(4) Possibility of modifications of the bound substrate. (5) The peptide chain is transferred from
the terminal PCP domain onto the TE domain via a transesterification reaction. (6) Final product
is released by hydrolysis or macrocyclization. The number of modules and modification
domains varies with each specific multienzyme system (Strieker et al., 2010).
1.4.1 Assembly logic of NRP synthesis
A typical NRPS consists of an initiation, extension and termination module (Figure 1.6). The
minimal NRPS module consists of two catalytic domains and a carrier protein, which together
carry out the selection and activation of the substrate and formation of the peptide bond. These
core domains are the A, C and PCP (otherwise known as the thiolation (T) domain), which
appear in the canonical order C-A-PCP(T) (Figure 1.5). The initiation module does not contain
Chapter 1 - Introduction
21
a C domain (Finking & Marahiel, 2004; Mootz et al., 2002). A fourth domain, a thioesterase
(TE), is often found at the C-terminus of the NRPS termination module and catalyzes the
release of the peptide from the NRPS (Felnagle et al., 2008).
Furthermore, in addition to the required domains involved in constructing the peptide
backbone, modules can contain a number of embedded editing/tailoring domains which are
able to create structural diversity by modifying the incorporated amino acid. These auxiliary
domains can act in cis or in trans during the biosynthetic process and examples include
epimerization (E), heterocyclization (Cy), oxidation (Ox), reduction (RE), N-methyltransferase
(MT), communication-mediating (COM) and aminotransferase (AMT) domains (Finking &
Marahiel, 2004).
1.4.1.1 Activation by the Adenylation domain
The A domain is often referred to as the most important domain of each module and this is
because it contains the substrate recognition and ATP-binding sites, which are necessary for
the activation of the specific substrate amino acid and formation of its acyl adenylate through
ATP hydrolysis (Marahiel, 1997).
The A domain selects the cognate amino acid from the available pool of substrates and tethers
it to the PCP domain in a two-step process. Briefly, it first catalyzes the formation of an
aminoacyl-adenylate intermediate through consumption of ATP, which is co-ordinated to Mg2+
and releases inorganic pyrophosphate (PPi) (Figure 1.7). Subsequently, the A domain allows
the transfer of the activated acyl moiety onto the free thiol group on the phosphopantetheinyl
arm of the active holo-PCP domain, thereby releasing AMP and producing a carboxy-thioester-
bound intermediate that is both thermodynamically activated and kinetically labile (discussed
in the following section) (Fischbach & Walsh, 2006; Sieber & Marahiel, 2005; Mootz et al.,
2002).
Chapter 1 - Introduction
22
Figure 1.7 The formation of the aminoacyl-adenylate intermediate catalysed by the A domain, at the
expense of ATP (adapted from Marahiel, 2009).
The A domain belongs to the large superfamily of adenylate-forming enzymes, which includes
all A domains of modular peptide synthetases and distinct proteins such as acetyl-CoA
synthetases, luciferases, oxidoreductases and 4-coumaryl CoA ligases. All of these enzymes
possess the ability to activate their carboxylic acid or amino acid substrates to their acyl
adenylates at the expense of ATP (Konz & Marahiel, 1999; Marahiel, 1997). They all share a
homologous domain of approximately 550 amino acids that consists of a set of highly
conserved signature sequences, which have been shown to be the major determinants of the
substrate specificity exhibited by A domains (Marahiel, 1997).
The determination of the crystal structures of two members of this adenylate-forming enzyme
superfamily, firefly luciferase of Photinys pyralis and the phenylalanine-specific A domain of
the gramicidin S synthetase (GrsA) of Bacillus brevis, termed PheA, provided fundamental
insight into the structural basis of substrate recognition and activation (Finking & Marahiel,
2004; Stachelhaus et al., 1999; Conti et al., 1997; Marahiel, 1997). Furthermore, it was
observed that even though the protein sequence similarity between PheA and the firefly
luciferase is only 16 %, the enzymes share a highly conserved three-dimensional structure. It
can be expected that very similar three-dimensional structures would be observed for all A
domains of peptide synthetase origin that show 30-60 % sequence identity to PheA (Marahiel,
1997).
The A domain of GrsA folds into a large amino-terminal and a smaller carboxy-terminal
subdomain. An ordered layer of water molecules mediate interactions between the two sub-
Chapter 1 - Introduction
23
domains. Ten highly conserved core motifs which surround the active site where the substrate
binds are associated with the amino-terminal domain, however, a lysine residue located in the
carboxy-terminal domain provides an essential interaction in co-ordinating the α-carboxyl
group of the substrate amino acid (Sieber & Marahiel, 2005; Stachelhaus et al., 1999).
The conserved motifs serve as functional anchors and it has been shown that many of the amino
acid residues within these motifs perform a key role in the co-ordination of ATP binding and
hydrolysis, as well as adenylation of the specific substrate carboxy moiety (Sieber & Marahiel,
2005; Stachelhaus et al., 1999). It has, however, been observed that some A domains display
“relaxed” substrate specificity and in these cases, chemically or sterically similar amino acids
are also recognised and analogously processed (Keating & Walsh, 1999).
1.4.1.2 Transport of substrates and intermediates to the catalytic centres by the
Peptidyl Carrier Protein
Located downstream of the A domain, the equally important PCP domain is approximately 80-
100 amino acid residues in length and is highly homologous to the acyl carrier protein (ACP)
involved in fatty acid synthases (FASs) and polyketide synthases (PKSs) (Sieber & Marahiel,
2005). Carrier proteins, also referred to as thiolation domains, may either be freestanding or
embedded in enzymatic systems. A variant of the carrier protein commonly found in
siderophore NRPS systems, is the aryl carrier protein (ArCP) (Qiao et al., 2007). The carrier
proteins are tasked with keeping reaction intermediates bound to the enzymatic machinery and
are responsible for transportation of substrates and elongation intermediates to the catalytic
centres of the PKS, NRPS or FAS assembly lines (Qiao et al., 2007; Stachelhaus et al., 1996).
In order for the PCP to become functional, each inactive apo-PCP domain must be post-
translationally modified (“primed”) by a phosphopantetheinyl transferase (PPTase), which
involves the transfer of the cofactor, 4’-phosphopantetheine (4’-PP), from coenzyme A (CoA)
to a conserved serine residue on the PCP domain. The PPTase stimulates the nucleophilic
attack of the PCP serine hydroxyl group on the pyrophosphate bridge of CoA, resulting in the
transfer of 4'-PP to the PCP domain and release of 3',5'-ADP (Lambalot et al., 1996). This, in
turn, results in an active holo-PCP domain with a flexible phosphopantetheinyl arm that is able
to covalently bind both the amino acyl and peptidyl substrates as energy-rich thioesters (Figure
Chapter 1 - Introduction
24
1.8) (Fischbach & Walsh, 2006; Schwarzer et al., 2003). The PCP-domain is then able to act
as the transport unit which enables the activated amino acids and elongation intermediates to
move between all of the catalytically active entities, as the A and C domains associate closely
to form a catalytic platform onto which the PCP domain is flexibly tethered between their active
sites (Tanovic et al., 2008).
Figure 1.8 Modification of the PCP domain by a phosphopantetheinyltransferase (PPTase). The transfer
of the 4’- phosphopantetheine cofactor from coenzyme A to a conserved serine residue in the
PCP domain is catalysed by members of this enzyme class (adapted from Fischbach & Walsh,
2006).
1.4.1.3 Peptide elongation by the Condensation domain
The C domain is approximately 450 amino acids in length and is responsible for the elongation
of the peptidyl chain. C domains are normally localized between each consecutive A-PCP
domain and catalyse the condensation reaction between the peptidyl chain tethered to the
phosphopantetheinyl arm of the upstream PCP domain and the amino acid bound to the
downstream PCP domain (Lautru & Challis, 2004). However, in the first condensation reaction
of an NRPS, both these reaction intermediates would typically be aminoacyl groups attached
to their respective PCP domains. Furthermore, C domains are absent from modules involved
in peptide initiation (Mootz et al., 2002).
Chapter 1 - Introduction
25
There is little information concerning the exact mechanism of peptide elongation and how the
interaction of modules affects the direction of polymerization, but it is thought that peptide
bond formation proceeds via the nucleophilic attack of the free α-amino group on the
downstream PCP-bound acceptor amino acid on the activated carboxy-thioester of the
upstream PCP-bound donor amino acid (Figure 1.9). This reaction facilitates the translocation
of the growing peptide chain onto the next module for elongation and structural changes
(Bloudoff et al., 2013; Sieber & Marahiel, 2005; Mootz et al., 2002; Marahiel, 1997).
Moreover, this condensation reaction is strictly unidirectional, leading to a downstream-
directed synthesis of the NRPS product (Samel et al., 2007).
Figure 1.9 Peptide elongation catalysed by the C domain, which involves an attack of the nucleophilic
amine of the acceptor substrate onto the electrophilic thioester of the donor substrate (Sieber &
Marahiel, 2005).
In order to shed light on the catalytic mechanism of the C domain, three structures that contain
NRPS C domains have been determined by X-ray crystallography, including a stand-alone C
domain (Keating et al., 2002), a C-PCP didomain complex (Samel et al., 2007) and a C-A-
PCP-TE termination module (Tanovic et al., 2008). The overall architecture of the C domain
revealed a pseudodimeric configuration consisting of both an N-terminal and C-terminal
subdomain. The active site is located at the bottom of a “canyon” or “V-shape” formed by the
two subdomains and is covered by a “latch” that crosses over from the C to N subdomain
Chapter 1 - Introduction
26
(Figure 1.10) (Bloudoff et al., 2013; Marahiel, 2009; Finking & Marahiel, 2004). The catalytic
centre includes a conserved HHxxxDGxS core motif (where x is any amino acid and defined
residues are represented by the single-letter amino acid code), that is also found in
dihydrolipoyl transacetylases, chloramphenicol acetyltransferases, and NRPS epimerization
and Cy domains (Sieber & Marahiel, 2005; Keating & Walsh, 1999; Marahiel et al., 1997). A
model, supported by mutational studies, suggests that the second histidine of this motif, which
is located at the bottom of the canyon, may act as a catalytic base promoting the deprotonation
of the NH3+ moiety of the thioester-bound nucleophile prior to peptide bond formation.
However, recent pK value analysis of this active site residue suggests that peptide bond
formation may depend mainly on electrostatic interactions rather than a general acid/base
catalysis (Marahiel, 2009; Samel et al., 2007; Konz & Marahiel, 1999).
Figure 1.10 Structure of the PCP-C didomain from the surfactin synthetase illustrating the active site
histidine (His) residue located on the floor of the C-domain “canyon” (Marahiel, 2009).
Chapter 1 - Introduction
27
1.4.1.3.1 Heterocyclization domains
The C domain of a module, which catalyzes basic peptide bond formation only, can be replaced
by a specialised heterocyclization (Cy) domain that shares striking structural and functional
homology to the C domain. The Cy domain combines the condensation function of the C
domain with additional heterocyclization and dehydration functions using the side chains of
the amino acids cysteine, serine or threonine within the product peptide backbone to produce
thiazoline (from cysteine), oxazoline (from serine) or 5-methyloxazoline (from threonine)
heterocycles (Walsh et al., 2001).
Cy domains were first identified in 1997 in the cyclic dodecylpeptide antibiotic bacitracin
synthetase, produced by Bacillus licheniformis ATCC 10716 (Konz et al., 1997) and were
further validated in the biochemical characterisation of yersiniabactin, an iron-chelating
virulence factor of Yersinia pestis (Gehring & Walsh, 1998). The catalytic core motif,
HHxxxDGxS, of the C domain is modified to DxxxxDxxS in the Cy domain. The conserved
aspartate residues are critical for both condensation and heterocyclization (Keating et al., 2002;
Keating & Walsh, 1999). Examples of secondary metabolites produced using Cy domains are
epothilone A, myxothiazol and mycobactin A produced by Sorangium cellulosum So ce90,
Stigmatella aurantiaca DW4/3-1 and M. tuberculosis, respectively (Figure 1.11) (Duerfahrt et
al., 2004).
Chapter 1 - Introduction
28
Figure 1.11 Heterocyclic ring-containing secondary metabolites from various organisms (the five-
membered thiazole and oxazole rings are shaded grey) (Duerfahrt et al., 2004).
The first reaction step catalysed by Cy domains is peptide bond condensation, carried out by
the nucleophilic attack of a PCP-bound cysteine, serine or threonine acceptor substrate onto
the thioester of the donor substrate. The next step involves the nucleophilic attack by the
hydroxyl side chain of serine/threonine or thiol side chain of cysteine onto the carbonyl C atom
of the newly-formed peptide bond to yield hemiaminal or thiohemiaminal intermediates and
the five-membered heterocyclic ring. The intermediates are subsequently dehydrated to yield
the C=N bond and the final oxazoline or thiazoline product (Figure 1.12) (Sieber & Marahiel,
2005; Walsh et al., 2001). In non-ribosomal peptide products such as the glycopeptide
antibiotic, bleomycin (produced by ‘Streptomyces verticillatus’ ATCC 15003) or myxothiazol,
additional oxidation (Ox) domains convert these heterocycles into more stable oxazole or
thiazole rings. Heterocyclic rings are common structural features of NRPs and are important
for the interaction with proteins, DNA and RNA, as well for chelating metal ions (Walsh,
2004).
Chapter 1 - Introduction
29
Figure 1.12 The formation of thiazoline (top of figure) and oxazoline heterocycles (bottom of figure) from
cysteine and threonine precursors, respectively (Walsh et al., 2001).
1.4.1.4 Peptide release by the Thioesterase domain
The termination of peptide synthesis on an NRPS assembly line is catalysed by the terminal
enzyme of the last module. During synthesis, the growing peptide chain is transported from
one module to the next until it reaches the final module’s PCP domain. Product release is then
achieved by the catalytic action of a C-terminal TE domain, which occurs when the nascent
peptidyl chain is transferred from the terminal PCP to a highly conserved active site serine
residue present in the TE domain to generate a covalent acyl-enzyme intermediate. This
intermediate can then be released by the external nucleophile, water, in a hydrolysis reaction
to form a linear acid or by the predominant mechanism of regio- and stereoselective
intramolecular macrocyclization using an internal nucleophile to produce a cyclic peptide
(Figure 1.13) (Sieber & Marahiel, 2005; Mootz et al., 2002). Macrocyclization is believed to
be favoured due to the fact that cyclic products are more resistant to proteolytic cleavage. In
Chapter 1 - Introduction
30
addition, TE domains that catalyse a cyclization reaction are also referred to as peptide cyclases
(Marahiel, 2009; Sieber & Marahiel, 2005; Finking & Marahiel, 2004).
The crystal structures of several dissected TE domains has revealed the presence of the
common fold of α/β-hydrolases, such as lipases and esterases, as well as the fact that the active
site consists of a catalytic triad composed of serine at position 80 (Ser80), histidine at position
207 (His207) and aspartate at position 107 (Asp107). Within this triad, the serine residue
serves as the site of tetrahedral intermediate formation that is stabilized by an oxyanion hole
en route to the acyl-enzyme intermediate. This intermediate is attacked by the action of an
internal nucleophile of the peptide chain (which can be the N-terminal amino group or a
functional side chain) or water, which results in a macrocyclic product or linear peptide,
respectively. It remains uncertain as to how the active site is sufficiently sealed from water in
order to catalyse cyclization rather than hydrolysis. However, the structures of the TE domains
have also revealed a flexible “lid” region that may possibly adopt an open conformation for
substrate entry and a closed conformation for excluding water from the active site (Condurso
& Bruner, 2012; Marahiel, 2009; Schwarzer et al., 2003).
Figure 1.13 Illustration depicting peptide release by the TE domain. Product release can be achieved either
by the external nucleophile, water, to produce a linear acid product as observed in (A) or by an
internal nucleophile to produce a cyclic product as observed in (B) (Sieber & Marahiel, 2005).
Chapter 1 - Introduction
31
1.4.1.5 Editing/tailoring domains
In addition to the essential domains found in every NRPS, further modifications to the final
product can be achieved by the action of several editing/modifying domains that act to increase
the diversity of NRPs. These so-called modifying domains are not present in every NRPS
system, but are nevertheless vital for the proper processing of their designated substrate within
their respective synthetase. Inactivation or deletion of these domains usually results in the
synthesis of products with severely reduced or no bioactivity.
1.4.1.5.1 Methylation
Certain non-ribosomal peptides such as cyclosporin, enniatin, actinomycin and yersiniabactin
possess N-methylated or C-methylated peptide bonds. These modifications are introduced by
a methyltransferase (MT) domain, embedded within the canonical fold of an associated A
domain, thus making the peptide less susceptible to proteolytic degradation. The MT domain,
including both N- and C-methyltransferases, is able to catalyse the transfer of the S-methyl
group from S-adenosylmethionine (SAM) to the α-amino group of the thioesterified aminoacyl
intermediate in C-A-MT-PCP modules, thereby releasing S-adenosylhomocysteine as a
reaction byproduct (Figure 1.14) (Fischbach & Walsh, 2006; Sieber & Marahiel, 2005; Finking
& Marahiel, 2004).
Figure 1.14 The MT domain catalyzes the transfer of the CH3 (Me) group from SAM to the amino group
of the aminoacyl-S-intermediate prior to peptide formation with the upstream peptidyl chain
(Walsh et al., 2001).
Chapter 1 - Introduction
32
1.4.1.5.2 Epimerization (control of stereochemistry)
A prevalent structural feature of NRPs is the incorporation of the less common D-amino acids.
Two different strategies are utilized by NRPSs for their incorporation, which may be
accomplished simply by an A domain with exclusive specificity towards a D-amino acid or by
epimerization of L-amino acids by integrated epimerization (E) domains (Sieber & Marahiel,
2005). The E domains are found on the carboxy terminus of the respective module’s PCP
domain and catalyse the racemization of the PCP-bound amino acid or of the C-terminal amino
acid of the growing peptidyl chain. Two different types of E domains catalyse these reactions
and are known as either aminoacyl epimerases, which can only be part of initiation modules,
or peptidyl epimerases, which are part of elongation modules. Due to the fact that E domains
are only involved in catalyzing the racemization of their substrates, specific incorporation of
the D-amino acid into the growing peptide chain is achieved by the enantiomer-selective donor
site of the downstream C domain. The C domain therefore acts as a type of “filter”, which
selectively withdraws the correct enantiomer from the pool of L/D-amino acids (Schwarzer et
al., 2003). In some rare cases, such as in the arthrofactin synthetase found in Pseudomonas sp.
MIS38, the C domain itself also exhibits epimerization activity in addition to its normal
function and it is then known as a “dual C/E” domain (Balibar et al., 2005).
1.4.1.5.3 Reduction domain
A reduction (RE) domain replaces the TE domain in a few NRPS systems, such as in the
biosynthesis of gramicidin A in B. brevis and safracin in Pseudomonas fluorescens. RE
domains, such as those found in the biosynthesis of myxochelin A in Angiococcus disciformis,
catalyse an alternative mechanism of peptide release that involves the NADPH-dependent
reduction of the PCP-bound peptidyl thioester to yield an unstable thioacetal intermediate that
is converted into a linear aldehyde or alternatively into an alcohol via a second reduction of the
thioacetal group (Figure 1.15) (Velasco et al., 2005; Gaitatzis et al., 2001). Another rare type
of chain termination strategy is found in some NRPSs, where the RE domain triggers the
reductive release, via nucleophilic attack by the free N-terminal amino group, of a highly
reactive peptide aldehyde that results in the subsequent formation of a stable macrocyclic imine
(Kopp & Marahiel, 2007). RE domains are also known to catalyse the NADPH-dependent
Chapter 1 - Introduction
33
reduction of thiazoline into thiazolidine by the addition of two electrons, as witnessed in one
of the rings in yersiniabactin (Sieber & Marahiel, 2005; Velasco et al., 2005).
Figure 1.15 The RE domain reduces the C-terminal carboxy-group to an aldehyde or to the corresponding
alcohol using NADPH as cofactor (Schwarzer et al., 2003).
1.4.1.5.4 Oxidation domain
Oxidation (Ox) domains often appear in the same module as a Cy domain in order to catalyze
the oxidation of the hydrolytically labile dihydroheterocyclic thiazolines and oxazolines by
two-electron transfer to yield stable heteroaromatic thiazole or oxazole rings (Figure 1.16)
(Duerfahrt et al., 2004; Schwarzer et al., 2003). Ox domains can be localized in the
accompanying A domain, as is the case in the epothilone synthetase, or they can be
C-terminally fused to the PCP domain of the respective module, as witnessed in the bleomycin
synthetase (Schwarzer et al., 2003; Du et al., 2000). Furthermore, flavin mononucleotide
(FMN) has been verified as a cofactor of Ox domains. FMN is reduced to FMNH2 during the
oxidation of the substrate and is subsequently re-oxidised by the reduction of molecular oxygen
to superoxide. While the oxidation of thiazoline to thiazole in the epothilone synthetase is
catalysed in cis by an FMN-containing Ox domain (a corresponding in cis flavoprotein domain
exists in the bleomycin synthetase), a second trans-acting Ox domain has been proposed to be
involved in the formation of the second thiazole ring found in the structure of bleomycin A2
(Finking & Marahiel, 2004; Du et al., 2000). The fact that these enzymes are able to act in
trans, where they are able to recognise the peptidyl chains on NRPS modules by protein-protein
Chapter 1 - Introduction
34
interactions, is testament to the enormous structural variation created in these natural products
(Walsh et al., 2001).
Figure 1.16 FMN-containing Ox domains catalyze the two electron oxidation of thiazolines and oxazolines
to yield stable heteroaromatic thiazole or oxazole rings, respectively (Schwarzer et al., 2003).
1.4.1.5.5 Further modifications
In the case of the linear gramicidin A, the N-terminus of the non-ribosomal peptide possesses
a formyl group which is introduced by a formylation (F) domain, localized N-terminal to the
A domain of the initiation module. The F domain is responsible for the N-formylation reaction
by means of the cofactor N-formyltetrahydrofolate. Other formylated NRPs include
coelichelin, whose ornithine residues are believed to be N-formylated in trans and
anabaenopeptilide 90-A whose N-terminus exhibits an N-formylated glutamine residue (Lautru
et al., 2005; Sieber & Marahiel, 2005).
Surfactin and fengicin are lipopeptides, which represent a subgroup of NRPs whose peptide
backbones are N-terminally bound to a fatty acid. An N-terminal C domain, in addition to the
A and PCP domain, is usually present in the initiation module of these NRPSs and is thought
to catalyse bond formation to the fatty acid.
An aminotransferase (AMT) domain exists within the mycosubtilin NRPS that converts a
thioesterified β-ketoacyl intermediate to a covalently tethered β-aminoacyl thioester with
Chapter 1 - Introduction
35
simultaneous removal of the α-amino group of glutamine to form α-keto-glutamine in a
variation of the classic transamination reaction. It was therefore proposed to be responsible for
the transfer of an amine group to the β-position of the growing acyl chain. The ability to
incorporate amine functionality directly into NRPs creates a functional handle for
macrocyclization strategies and acts as a potent hydrogen bond donor, which can significantly
alter the biology of a given compound (Fischbach & Walsh, 2006).
All of the modifications that have been introduced thus far are catalysed by domains that are
embedded into their respective NRPS modules and almost entirely affect only the peptide
backbone. Additionally, certain enzymes such as glycosyl transferases, halogenases and
hydroxylases are able to modify the peptide’s side chains and are generally encoded in the same
biosynthetic gene cluster.
1.4.2 NRPS substrate specificity prediction and the non-ribosomal code
The A domain has been described as the most important domain within each NRPS module
due to the fact that it recognizes and activates only the cognate substrate amino acid as its acyl
adenylate (using ATP to drive the reaction). Hence A domains act in a ‘lock and key’ process
and are considered the primary determinant of substrate specificity, thereby also defining what
molecules can be synthesized by the specific NRPS (Lautru & Challis, 2004; von Döhren et
al., 1999). Knowledge about how A domains are able to recognize their specific substrates is
of fundamental importance to understanding and manipulating these complex enzymatic
systems for use as scaffolds for combinatorial biosynthesis. Combinatorial biosynthesis
(section 1.4.4) could allow for the generation of novel derivatives of the non-ribosomal peptide
and ideally new, more potent antibiotics (Rausch et al., 2005; Lautru & Challis, 2004; Challis
et al., 2000; Conti et al., 1997).
The study of A domain specificity was greatly facilitated by the determination of the crystal
structure of firefly luciferase and the phenylalanine activating domain, PheA, from the first
module of gramicidin synthetase (GrsA). The co-crystalization of PheA in a ternary complex
with L-phenylalanine (L-phe) and AMP allowed the discovery of ten highly conserved motifs,
denoted A1 to A10, which extend over a region of 450 amino acids and mostly surround the
active site where the substrate binds. The identification of this hydrophobic L-phe binding
Chapter 1 - Introduction
36
pocket and the A domain residues making contact with L-phe provided a structural basis for
understanding the specificity of peptide synthetases such as NRPSs (Rausch et al., 2005; Lautru
& Challis, 2004; Challis et al., 2000; Conti et al., 1997).
Expanding on the approach pursued by Conti and colleagues, Stachelhaus et al. (1999) and
Challis et al. (2000) compared the L-phe binding pocket of GrsA with the corresponding
sequences of over 150 aminoacyl and iminoacyl adenylate-forming domains of NRPSs to
pinpoint the essential amino acid residues involved in substrate specificity and binding. Both
studies examined a ~100 amino acid stretch between the core motifs A4 and A5 and identified
10 amino acid residues that were within approximately 5.5 Å of the active site-bound
phenylalanine and in contact with the substrate (Rausch et al., 2005; Lautru & Challis, 2004;
Conti et al., 1997).
These 10 amino acid residues were determined to be crucial for substrate binding and catalysis,
as alignments of consensus A domain residues to the PheA domain revealed that all A domains
display the same type of binding pockets, with different key residues that interact with different
amino acid substrates. It was postulated that two residues, Asp235 and Lys517, stabilize the α-
amino group of the amino acid substrate via two hydrogen bonds and are critical for the correct
positioning of the substrate within the active site for ATP-dependent activation. These two
residues are located in the conserved core motifs of A4 (Asp235) and A10 (Lys517). The other
residues bordering the PheA specificity binding pocket were determined to be Ala236, Ile330
and Cys331 on the one side and Ala322, Ala301, Ile299 and Thr278 on the other side of the
pocket. Both sides are separated by the indole ring of tryptophan at position 239 (Trp239),
which is located at the bottom of the pocket (Stachelhaus et al., 1999).
More importantly, it was determined that due to the high degree of sequence identity shared
between NRPS A domains, the amino acid residues that correspond to those lining the PheA
binding pocket could be used to reveal substrate specificity in other A domains. The
consecutive order of the 10 residues was determined to constitute the signature sequence
involved in the binding pockets of A domains and can be interpreted as the “specificity-
conferring code” (also referred to as the non-ribosomal code), as it allows for the prediction of
A domain selectivity on the basis of the A domain primary sequence (Figure 1.17) (Table 1.1)
(Rausch et al., 2005; von Döhren et al., 1999).
Chapter 1 - Introduction
37
Figure 1.17 A multiple sequence alignment of the primary amino acid sequences from known A domains in
order to determine the selectivity-conferring residues (a). The sequence of ~100aa between the
core motifs A4 and A5 from PheA from GrsA was aligned with the corresponding sequence of
AspA from the surfactin synthetase, SrfA, OrnA from the gramicidin synthetase, GrsB3 and
ValA from the cyclosporine synthetase. Yellow residues indicate those involved in the binding
pocket positions and brown residues indicate conserved motifs which anchor the alignment. (b)
Ten highly conserved residues were extracted from the sequence alignment and the consecutive
order of the amino acids was determined to constitute the signature sequence involved in the
binding pockets of the aligned A domains. The missing residue, Lys517, is highly conserved
within motif A10, which is not shown in the protein sequence. The alignment was extended to
160 different A domains to confirm accuracy in determining the signature sequence (adapted
from Stachelhaus et al., 1999).
This discovery confirmed previous findings from site-directed mutagenesis and photoaffinity
labelling experiments that indicated that these amino acid residues were involved in ATP
binding and hydrolysis (Gocht & Marahiel, 1994; Pavela-Vrancic et al., 1994). The non-
ribosomal code was initially restricted to amino acid-activating A domains but was extended
to carboxy acid-activating A domains when the crystal structure of the stand-alone 2,3-
dihydroxybenzoic acid activating domain, DhbE, from Bacillus subtilis was solved (May et al.,
2002).
Chapter 1 - Introduction
38
Table 1.1 Consensus specificity code for substrates from several adenylation domains
Clusters of signature sequences extracted from A domains activating the same substrates were used to determine
the consensus sequences for the recognition of several amino acid substrates. The biosynthetic template from
which each A domain specificity code was derived is included, along with the overall similarity of each signature
sequence. Variable constituents within each codon are represented by red residues and ‘wobble’-like positions,
which reveal a large degree of variability throughout all codons, are indicated in blue. Aad, δ(L-α-aminoadipic
acid); Dab, 2,3-diamino butyric acid; Dhb, 2,3-dihydroxy benzoic acid; Sal, salicylate; Phg, L-phenylglycine;
hPhg, 4-hydroxy-L-phenylglycine; Pip, L-pipecolinic acid; Dht, dehydrothreonine; ‘@’ indicates a modification
of the residue (Stachelhaus et al., 1999).
Chapter 1 - Introduction
39
The successful mutation of all the key residues of the PheA binding pocket, which resulted in
relaxation or alteration of its substrate specificity, demonstrated the reliability of the non-
ribosomal code. This has resulted in the development of web-based NRPS prediction services
such as the NRPS-PKS knowledge base (Ansari et al., 2004), NP.searcher (Li et al., 2009),
PKS/NRPS analysis (Bachmann & Ravel, 2009) and antiSMASH 3.0 (Weber et al., 2015),
which make use of the “specificity-conferring code” to predict putative A domain substrates in
NRPS genes. Although these tools have been relatively successful at predicting the specificities
of A domains in new NRPS genes, they do have a number of weaknesses (Challis et al., 2000).
A clear shortcoming is that predictions of substrate specificities are based on known A domain
sequences. Since not all A-domain sequences in nature are known and, in particular, since there
are relatively few sequences for A domains that bind more unusual substrates, the accuracy of
substrate specificity prediction is limited. The specificity of uncharacterised A domains must
therefore be deduced from the available code for domains with known specificity. It has been
observed that there are deviations from the code. For example, not all A domains specific for
phenylalanine have the exact same specificity binding pocket sequence as GrsA, e.g. BarG of
the barbamide synthesis gene cluster from Lyngbya majuscula (GenBank accession number:
AAN32981) has DAWTVAAVCK instead of DAWTIAAICK. In addition, there are examples
of codes where the predicted substrate specificity does not correspond to the actual activated
amino acid, such as in the alanine-activating domain Sare0718 from the marine actinomycete
Salinispora arenicola CNS-205 (which has the code for valine) and in the biosynthetic cluster
for fusaricidin, a mixture of 12 depsipeptides from Paenibacillus polymyxa PKB1, which
displays relaxed substrate specificity and allows for the incorporation of D-amino acids instead
of their L-isomers (Xia et al., 2012; Li & Jensen, 2008).
A further shortcoming has been observed in the analysis of the active sites within the A domains
of certain types of NRPSs, particularly those belonging to fungi, as the GrsA crystal (from a
bacterium) seems to be an inadequate model for fungal NRPSs or because the large number of
sequence variants in the active site of fungal NRPSs does not allow for the identification of the
key residues required for substrate-specificity prediction (Prieto et al., 2012). Certain positions
are considered more variable or ‘wobble’-like than others, particularly 239, 278, 299, 322 and
331, which are highly variant. Furthermore, positions 235 and 517 are considered invariant
and positions 236, 301 and 330 are moderately variant (Table 1.1). The variability reflects
each position’s importance in contributing to substrate specificity, but also causes dissimilarity
Chapter 1 - Introduction
40
between the signature sequences for A domains specific for identical substrate amino acids
(Stachelhaus et al., 1999).
It is due to these limitations that Rausch et al. (2005) expanded on the work of Stachelhaus et
al. (1999) and Challis et al. (2000) by publishing a machine learning algorithm in which
transductive support vector machines (TSVMs) were utilised to statistically propose NRPS A
domain specificity using the physico-chemical fingerprint of the residues within 8 Å of the
active site of the A domain (a total of 34 residues). The residues are encoded into TSVMs
based on their physico-chemical properties such as the number of hydrogen bond donors,
polarity, volume, secondary structure preferences, hydrophobicity and isoelectric point and a
continuously updated dataset of A domains with known specificity are used in predicting
substrate specificity. Due to the occurrence of relaxed/promiscuous specificity in certain A
domains, such as in the NRPS responsible for xenematide biosynthesis in Xenorhabdus
nematophila, the specificities for substrates with similar physico-chemical properties are
clustered together (Crawford et al., 2011; Rausch et al., 2005).
An open source web-based predictor, NRPSpredictor, based on TSVMs, was built on the 34
active site residues in order to predict A domain specificity, which was later refined and
replaced by the NRPSpredictor2, which possesses improved prediction performance, two new
prediction levels and a larger database (Rottig et al., 2011).
Although the TSVM-based method was based on a more recent database and was able to
provide a substrate-specificity prediction in an additional 18% of cases, it is still beneficial to
use it in combination with the empirical predictive method developed by Stachelhaus et al.
(1999) and Challis et al. (2000) in order to create an even more accurate prediction tool (Rausch
et al., 2005). This is largely due to the fact that most of the weaknesses of the older method
remain and by expanding the number of residues considered, it may have amplified the
problems associated with including data with little or no influence on the specificity of the A
domains. In addition, the clustering of specificities reduces the accuracy of the predictions
and, although this is acceptable for A domains which possess relaxed specificity, other A
domains are much more specific. There is currently no way to distinguish a highly specific A
domain from a relaxed one (Rausch et al., 2005; Mootz et al., 2002; Challis et al., 2000).
Chapter 1 - Introduction
41
This situation prompted interest in developing new prediction methods supported by other
approaches, such as the use of hidden Markov Models (HMM). Khurana et al. (2010) applied
HMM to functionally classify the acyl-CoA synthetase superfamily members. The results of
this work suggest that the application of HMM to classify this superfamily outperforms the
predictions based on a restricted number of active site residues (Khurana et al., 2010).
Furthermore, a novel two-mode factor analysis model based on latent semantic indexing (LSI)
has recently been published by Baranasic et al. (2013). This model is able to predict the specific
amino acid that is activated by the A domain in contrast to a cluster of similar amino acids.
The authors suggest that a detailed comparison of prediction quality against those of the
NRPSpredictor, showed that the LSI model performed slightly better and is thus the most
accurate method currently available for prediction of A domain substrate specificities
(Baranasic et al., 2013).
1.4.3 NRPS biosynthetic strategies
The order in which the modules of an NRPS are utilised to construct the final product and the
assembly of the core domains within each module can vary considerably, thereby allowing
these enzymatic systems to synthesize diverse chemical structures. The three most common
biosynthetic strategies employed by NRPSs include the linear or type A NRPS, iterative or
type B NRPS and the nonlinear or type C NRPS (Mootz et al., 2002).
The simplest biosynthetic strategy is that of the linear or type A NRPSs, in which the three core
domains are arranged in the order C-A-PCP in an elongation module, which is able to add one
amino acid to the growing peptide chain (Figure 1.18). The initiation module is responsible
for incorporating the first amino acid of the peptide chain and does not contain a C domain,
whereas, the termination module contains a TE domain in order to catalyse the release of the
final product. Therefore, a typical, linear NRPS template consists of n modules with a domain
organization in the order of A-PCP-(C-A-PCP)n-1-TE, for construction of a final peptide chain
containing n amino acids. The sequence of the final peptide chain is determined exclusively
by the number and order of the modules, which differs in the case of nonlinear and iterative
NRPSs. Examples of linear NRPSs include those involved in the biosynthesis of cyclosporin,
surfactin, actinomycin, peramine, ergovaline, tyrocidine and δ-(L-α-aminoadipyl)-L-cysteinyl-
D-valine (ACV), the isopenicillin and cephalosporin precursor (Mootz et al., 2002).
Chapter 1 - Introduction
42
Figure 1.18 Example of the module and domain organization, A-PCP-(C-A-PCP)n-1-TE, observed in a linear
or type A NRPS, such as the ACV synthetase, which synthesizes the isopenicillin precursor
ACV (Mootz et al., 2002).
In the iterative or type B NRPSs, modules are used more than once in an efficient method of
assembling multimeric peptide products. Instead of replicating the elongation modules, the
entire NRPS is used repeatedly in an iterative manner to construct peptide chains that consist
of recurring, short sequences. The product produced by the first cycle of synthesis is stalled
on the C-terminal TE domain, thereby regenerating the NRPS for the assembly of the next
product of the same amino acid sequence. Oligomerization of the completed product occurs
on the TE domain and is released via hydrolysis or macrocyclization, with the latter being the
preferred method. Examples of iterative NRPSs are those that synthesize enterobactin (Figure
1.19), gramicidin, enniatin and bacillibactin (Mootz et al., 2002).
The final biosynthetic strategy is known as the nonlinear or type C NRPS, which can, in most
cases, be identified from their primary sequence due to the fact that the identified module and
Chapter 1 - Introduction
43
domain organization will deviate from the classical C-A-PCP domain organization. They are
also characterized by their ability to incorporate small soluble molecules that are not covalently
tethered to the NRPS template during synthesis, such as the involvement of amines in
vibriobactin biosynthesis, instead of PCP-loaded amino acids (Finking & Marahiel, 2004;
Keating et al., 2002). Due to the fact that amines, such as norspermidine in vibriobactin
biosynthesis, lack a carboxyl group necessary for their covalent attachment as a thioester,
specialized C domains are employed to incorporate amines. The vibriobactin biosynthetic
cluster also encodes an unusual tandem arrangement of Cy domains, in which one is
responsible for heterocyclic ring formation, while the other’s function remains unclear (Mootz
et al., 2002). Furthermore, unusual internal cyclizations are often associated with deviations
from the standard domain organization observed in linear NRPSs, such as in the biosynthesis
of bleomycin (Mootz et al., 2002).
Other unusual biosynthetic strategies employed by type C NRPSs include utilizing a single
domain to catalyse multiple different reactions, such as the cysteine-specific A domain in the
yersiniabactin biosynthetic machinery, which is responsible for the loading of three different
PCPs (Suo et al., 2001), as well as the existence of a one-domain NRPS involved in the
biosynthesis of the antibiotic novobiocin in Streptomyces spheroides. The enzyme, NovL,
shares homology with acyl-adenylate forming enzymes of the same superfamily as A domains
and catalyzes formation of the peptide bond between 3-dimethylallyl-4-hydroxybenzoic acid
and 3-amino-4,7-dihydroxy-8-methyl coumarin in a reaction that is similar to those catalysed
by acyl-CoA ligases. NovL is able to activate the carboxy acid of 3-dimethylallyl-4-
hydroxybenzoic acid towards the acyl adenylate, but instead of transferring it onto a PCP
domain, the acyl adenylate acts as the electrophile for the condensation with the amino group
nucleophile of 3-amino-4,7-dihydroxy-8-methyl coumarin (Mootz et al., 2002). It is also
interesting to note that free-standing A domains are known to activate aromatic carboxylic
acids and transfer (acylate) them to ArCPs, which are fused to isochorismate lyase, an enzyme
involved in the synthesis of 2,3-dihydroxybenzoic acid, which is used as a starter unit in this
type of synthetase (Crawford et al., 2011; Schmoock et al., 2005).
Chapter 1 - Introduction
44
Figure 1.19 Example of the iterative or type B NRPS as observed in the biosynthesis of enterobactin. Three
Dhb-Ser-S-ppant intermediates are produced on the two modules of the enterobactin NRPS and
oligomerized and cyclized on the TE domain (Mootz et al., 2002).
Additionally, fragments of NRPS assembly lines such as A-PCP didomains or separate, but
adjacently encoded A and PCP domains are found in the absence of any other NRPS machinery
(Fischbach & Walsh, 2006; Ullrich & Bender, 1994). It was discovered that these A-PCP
didomains and freestanding A/PCP domains do in fact activate a specific L-amino acid as an
aminoacyl adenylate, which then acts as a substrate for a partner enzyme to chemically modify
the β-or γ-carbon of the thioesterified aminoacyl intermediate. Consequently, it has been
inferred that the use of didomains or freestanding domains is a strategy to sequester a portion
Chapter 1 - Introduction
45
of the pool of proteinogenic amino acids in order to modify them into a nonproteinogenic form,
which can be used in the biosynthesis of extremely diverse secondary metabolites (Fischbach
& Walsh, 2006).
The nonlinear NRPSs are impressive examples of how microbial producers are able to modify
assembly-line organizations and operations in order to evolve novel combinations of the
enzymatic components to generate new natural products (Fischbach & Walsh, 2006; Schwarzer
et al., 2003).
1.4.4 Combinatorial biosynthesis and intermolecular communication
Combinatorial biosynthesis can be defined as ”the application of genetic engineering to modify
biosynthetic pathways to natural products in order to produce new and altered structures using
nature’s biosynthetic machinery” (Floss, 2006). In this approach, which was first demonstrated
in work by David Hopwood and colleagues (Hopwood et al., 1985), biosynthetic genes are
cloned into an actinomycete host that does not naturally produce the antibiotic. Selected genes
in the gene cluster are then deleted or replaced with carefully-chosen antibiotic biosynthetic
genes from the producers of other actinomycete antibiotics, so that the host strain produces a
chemical derivative of the original antibiotic (i.e. a hybrid antibiotic), dictated by the
composition of the modified biosynthetic gene cluster (Weissman & Leadlay, 2005; Reynolds,
1998).
The order and domain composition of NRPS modules are the direct consequence of a
meticulous selection process during evolution to synthesize a peptide molecule with particular
bioactive properties. Once the logic and mechanisms of NRPS assembly had been investigated,
interest developed in the rational manipulation of the NRPS template to synthesize novel
peptide products (Sieber & Marahiel, 2005). Subsequently, over the past decade and a half,
several strategies have been developed to redesign NRPS assembly lines in order to generate
peptides with altered biological properties. Most of these exploits have focussed on the
combinatorial approach of swapping or deleting domains or modules, as well as the mutation
of individual domains to alter their specificity (Hahn & Stachelhaus, 2006). Although some of
the domain swapping experiments have been successful in the production of altered products,
a major stumbling block has been the fact that most of the chimeric NRPSs were functionally
Chapter 1 - Introduction
46
impaired and produced small amounts of the product (Giessen & Marahiel, 2012). However,
the identification of linker regions that bridge the individual modules within an NRPS and a
better understanding of the domain borders has allowed these approaches to remain viable. The
linker regions are about 15 amino acids in length and do not contain conserved residues. The
fact that these linker regions are able to connect individual modules, in the absence of any
conserved residues, has made them the ideal candidate for artificial module fusions. Indeed,
in vivo biochemical studies on the tyrocidin synthetase showed that the fusion of modules
within the linker regions resulted in the production of satisfying amounts of the altered peptide
product (Schwarzer et al., 2003).
Furthermore, the elucidation of the molecular basis for the selective interaction between NRPS
modules via communication-mediating (COM) domains has also widened the toolbox for the
combinatorial manipulation of NRPSs (Hahn & Stachelhaus, 2006). According to a study by
Hahn & Stachelhaus (2004), a donor COM domain located at the C terminus of an aminoacyl-
or peptidyl-donating NRPS and an acceptor COM domain located at the N terminus of the
accepting cognate NRPS form a compatible set, which facilitates the intermolecular
communication between adjacent modules. Recognition between the donor and acceptor
domains are mediated by electrostatic and/or polar interactions between five pairs of residues
located in both of the compatible COM domains. Additional studies have revealed that the
swapping of COM domains can be used to manipulate the interaction of enzymes within the
NRPS complex leading to the simultaneous production of different peptide products (Hahn &
Stachelhaus, 2006).
The recombination of whole modules does represent a rather drastic intervention in the
biosynthesis of NRPS-produced compounds, but eventually these approaches may lead to the
combinatorial biosynthesis of innovative peptide antibiotics. Combinatorial biosynthesis has
the potential to be a powerful way to generate antibiotic derivatives with greater antibacterial
activity and/or improved pharmacokinetic properties. It has advantages over the chemical
derivatisation of antibiotic molecules, since the bacterial biosynthetic enzymes carry out
site-specific and enantiomer-specific reactions, by-passing the need to protect reactive
functional groups in each reaction step in the chemical approach. This ensures that only the
desired product is produced, which enhances the product yield.
Chapter 1 - Introduction
47
1.5 RESEARCH AIMS
The actinomycete, Streptomyces polyantibioticus SPRT, was isolated from soil collected from
the banks of the Umgeni River, KwaZulu-Natal Province, South Africa, as part of an antibiotic-
screening programme (Le Roes-Hill & Meyers, 2009). It exhibits antibiosis against various
Gram-positive and Gram-negative bacteria, including Enterococcus faecium VanA (a
vancomycin-resistant strain), Mycobacterium aurum A+ (which has a similar antibiotic
susceptibility to M. tuberculosis, but is not pathogenic; Chung et al., 1995) and E. coli ATCC
25922 (Le Roes, 2005). Of great interest, however, was its antibiotic activity against M.
tuberculosis H37RvT, which prompted interest in its antibiotic production.
An antibacterial compound produced by S. polyantibioticus SPRT was isolated and its structure
was determined by X-ray crystallography and nuclear magnetic resonance (NMR) to be 2,5-
diphenyloxazole (DPO; Figure 1.20) (Le Roes, 2005). An independent report by Giddens et
al. (2005) confirmed that DPO (produced by chemical synthesis) showed activity against non-
replicating persistent cells of M. tuberculosis, which are difficult to eradicate using traditional
anti-TB drugs. The authors suggested that simple oxazole derivatives such as DPO may
therefore be feasible options in the search for new antitubercular agents. DPO is currently only
known to be synthesised chemically (Adrova et al., 1956), therefore its discovery from a
biological origin is of great interest. DPO is unusual in that it is a 2,5-disubstituted oxazole,
whereas most other disubstituted oxazoles from biological sources are 2,4-substituted.
Based on the structure of DPO, a biosynthetic scheme for the synthesis of this molecule was
proposed, whereby a NRPS condenses a molecule of benzoic acid with 3-
hydroxyphenylalanine. The resulting benzoyl-β-hydroxyphenylalanine is converted to DPO
by heterocyclisation, oxidation and decarboxylation.
It seems likely that DPO is synthesized non-ribosomally by S. polyantibioticus SPRT due to the
presence of an oxazole. The NRPS responsible for the biosynthesis of DPO is proposed to have
an A domain specific for the activation of phenylalanine or 3-hydroxyphenylalanine, plus
ArCP, Cy, Ox, PCP and TE domains.
Chapter 1 - Introduction
48
Figure 1.20 The structure of 2,5-diphenyloxazole (DPO).
The overall aim of this study was to confirm the production of DPO by S. polyantibioticus
SPRT and then to focus on the elucidation of the biosynthetic pathway involved in its synthesis.
This consisted of the identification and partial characterization of the genes involved in the
production of DPO to establish whether an NRPS is involved.
In order to determine whether the proposed biosynthetic pathway is correct, the genes coding
for benzoic acid synthesis and the DPO NRPS had to be identified in the S. polyantibioticus
SPRT genome. The aims of the study were thus:
(i) To identify the genes involved in joining benzoic acid and phenylalanine/3-
hydroxyphenylalanine in S. polyantibioticus SPRT to create a 2,5-disubstituted oxazole.
(ii) To identify the genes responsible for the biosynthesis of benzoic acid in S. polyantibioticus
SPRT.
(iii) To propose a pathway for the biosynthesis of DPO in S. polyantibioticus SPRT.
(iv) To develop a method to introduce recombinant plasmids into S. polyantibioticus SPRT.
Chapter 1 - Introduction
49
(v) To prove the involvement of genes in DPO biosynthesis through the creation of selected
gene knockouts in S. polyantibioticus SPRT.
Lastly, the genetic information from S. polyantibioticus SPRT will help to lay the foundation
for future combinatorial biosynthetic studies, using the genes responsible for DPO production
to develop a range of oxazole derivatives that could be tested for enhanced antitubercular
activity and therefore could become candidates for development as novel drugs to treat drug-
resistant tuberculosis.
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Waksman, S.A. (1947). What Is an Antibiotic or an Antibiotic Substance? Mycologia, 39: 565-569. Walsh, C.T. Chen, H., Keating, T.A., Hubbard, B.K., Losey, H.C., Luo, L., Marshall, C.G., Miller, D.A., & Patel, H.M. (2001). Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines. Current Opinion in Chemical Biology, 5: 525-534. Walsh, C.T. (2004). Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science, 303: 1805-1810. Walsh, C.T. & Fischbach, M.A. (2010). Natural Products Version 2.0: Connecting Genes to Molecules. Journal of the American Chemical Society, 132(8): 2469-2493. Wang, L., Yun, B., George, N.P., Wendt-Pienkowski, E., Galm, U., Oh, T., Coughlin, J.M., Zhang, G., Tao, M. & Shen, B. (2007). The Glycopeptide Antitumor Antibiotic Zorbamycin from Streptomyces flavoviridis ATCC 21892: Strain Improvement and Structure Elucidation. Journal of Natural Products, 70(3): 402-406. Watve, M.G., Tickoo, R., Jog, M.M., Bhole, B.D. (2001). How many antibiotics are produced by the genus Streptomyces? Archives of microbiology, 176(5): 386-90. Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H.U., Bruccoleri, R., Lee, S.Y., Fischbacj, M.A., Muller, R., Wohlleben, W., Breitling, R., Takano, E., & Medema, M.H. (2015). antiSMASH 3.0 — a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Research, doi: 10.1093/nar/gkv437 Weissman, K. J. & Leadlay, P. F. (2005). Combinatorial biosynthesis of reduced polyketides. Nature Reviews: Microbiology; 3: 925-936. World Health Organisation (WHO). (2014). Last accessed 12 January 2015. http://www.who.int/mediacentre/factsheets/fs104/en/. Wu, D., Hugenholtz. P., Mavromatis, K., Pukall, R., Dalin, E., Ivanova, N. N., Kunin, V., Goodwin, L., Wu, M., Tindall, B. J., Hooper, S. D., Pati, A., Lykidis, A., Spring, S., Anderson, I.J., D'haeseleer, P., Zemla, A., Singer, M., Lapidus, A., Nolan, M., Copeland, A., Han, C., Chen, F., Cheng, J.F., Lucas, S., Kerfeld, C., Lang, E., Gronow, S., Chain, P., Bruce, D., Rubin, E.M., Kyrpides, N.C., Klenk, H.P. & Eisen, J.A. (2009). A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature; 462: 1056-1060. Xia, S., Ma, Y., Zhang, W., Yang, Y., Wu, S., Zhu, M., Deng, L., Li, B., Liu, Z. & Qi, C. (2012). Identification of Sare0718 as an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205. PLoS One, 7(5): 1-12. Yeh, V.S.C. (2004). Recent advances in the total synthesis of oxazole-containing natural products. Tetrahedron 60: 11995 – 12042. Yoshioka, T., Mohri, K., Oikawa, Y. & Yonemitsu, 0. (1981). Metabolism of Streptoverticilium olivoreticuli and Streptomyces cinnamomeus. Journal of Chemical Research, 194. Young, D. B., Gideon, H. P. & Wilkinson, R. J. (2009). Eliminating latent tuberculosis. Trends in Microbiology, 17: 183-188.
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CHAPTER 2
EARLY ATTEMPTS AT ISOLATING THE GENE CLUSTER
RESPONSIBLE FOR DPO BIOSYNTHESIS IN STREPTOMYCES
POLYANTIBIOTICUS SPRT
60
CHAPTER 2
EARLY ATTEMPTS AT ISOLATING THE GENE CLUSTER
RESPONSIBLE FOR DPO BIOSYNTHESIS IN
STREPTOMYCES POLYANTIBIOTICUS SPRT 2.1 Abstract ........................................................................................................................ 62
2.2 Introduction .................................................................................................................. 64
2.2.1 Biosynthesis of DPO ........................................................................................ 64
2.2.2 Isolation of genes involved in DPO biosynthesis ............................................ 67
2.3 Materials and Methods ................................................................................................. 70
2.3.1 Strains, media and growth conditions .............................................................. 70
2.3.2 Genomic DNA extraction ................................................................................ 70
2.3.3 Primer design ................................................................................................... 70
2.3.4 PCR protocols .................................................................................................. 74
2.3.4.1 A domain amplification ........................................................... 74
2.3.4.2 Cy domain amplification.......................................................... 74
2.3.4.3 Ammonia lyase gene amplification ......................................... 75
2.3.4.4 paaK gene amplification .......................................................... 75
2.3.4.5 Long range PCR ....................................................................... 76
2.3.4.6 Colony PCR ............................................................................. 76
2.3.5 Splinkerette method ......................................................................................... 77
2.3.6 Cloning and sequencing ................................................................................... 78
2.3.7 Sequence analysis ............................................................................................ 79
2.3.8 Southern hybridization ..................................................................................... 80
2.3.8.1 Restriction endonuclease digestion .......................................... 80
2.3.8.2 Probe preparation ..................................................................... 80
2.3.8.3 Southern blot hybridization...................................................... 81
61
2.4 Results and Discussion ................................................................................................ 83
2.4.1 Isolation of the NRPS gene cluster .................................................................. 83
2.4.1.1 A domain amplification from S. polyantibioticus SPRT .......... 83
2.4.1.2 Southern hybridization using the phenylalanine-specific A
domain probe ........................................................................... 88
2.4.1.3 Cy domain amplification.......................................................... 89
2.4.1.4 NRPS domain amplification from Sts. Oxazolinicum.............. 92
2.4.2 Isolation of the genes involved in benzoic acid biosynthesis .......................... 92
2.4.2.1 Amplification of PAL/HAL ..................................................... 92
2.4.2.2 Southern hybridization using the encP gene probe .................. 94
2.4.2.3 Amplification of paaK ............................................................. 95
2.4.2.4 Southern hybridization using the paaK gene probe ................. 96
2.5 Conclusion ................................................................................................................... 98
2.6 Reference list ............................................................................................................... 99
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
62
CHAPTER 2
EARLY ATTEMPTS AT ISOLATING THE GENE CLUSTER
RESPONSIBLE FOR DPO BIOSYNTHESIS IN
STREPTOMYCES POLYANTIBIOTICUS SPRT
2.1 ABSTRACT
To determine whether the hypothesis pertaining to the DPO biosynthetic pathway is correct,
the genes coding for benzoic acid synthesis and the DPO NRPS in the S. polyantibioticus SPRT
genome need to be identified. As benzoic acid synthesis is unusual in bacteria, the initial focus
was on detecting an NRPS gene in S. polyantibioticus SPRT with an A domain exhibiting a
binding pocket substrate specificity for phenylalanine or 3-hydroxyphenylalanine, as this
would most likely indicate an NRPS involved in the biosynthesis of DPO. This first step was
achieved by performing PCR amplification of NRPS A domain sequences, which led to the
identification of 12 unique NRPS A domains. One of the A domains was specific for
phenylalanine. This phenylalanine–specific domain (designated A-18) was used as a probe in
Southern hybridization experiments in an effort to identify larger DNA fragments surrounding
the A-18 NRPS gene cluster. However, no further sequence information could be obtained.
The identification of Cy domains in the genome of S. polyantibioticus SPRT was analysed in a
similar fashion to the A domains, as the presence of a Cy domain would be an indicator of
heterocyclization across the amide bond, a reaction required for the formation of an oxazole
ring. PCR primers specific for oxazole- and thiazole-producing Cy domains were designed in
an effort to identify Cy-domain-containing NRPS genes in S. polyantibioticus SPRT. The Cy-
domain primers were unsuccessful. Similar attempts were made at identifying genes involved
in the synthesis of benzoic acid in S. polyantibioticus SPRT, as the presence of a phenylalanine
ammonia-lyase (PAL) encoding gene would allow the synthesis of benzoyl-CoA for DPO
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
63
production. PCR primers were utilised to amplify the encP gene (encoding PAL) from
'Streptomyces maritimus' DSM 41777T and Southern hybridization confirmed the presence of
encP in this strain. In contrast, an encP homologue could not be detected in the genome of S.
polyantibioticus SPRT. As S. polyantibioticus SPRT appears not to possess a PAL gene, it was
suggested that this organism may produce benzoyl-CoA via the phenylacetate (PA) pathway
involved in the degradation of phenylalanine. The initial step of the PA pathway is catalyzed
by a PA-CoA ligase and degenerate primers were used to amplify a homologue of the gene,
paaK, encoding this enzyme in S. polyantibioticus SPRT. The presence of a paaK gene in S.
polyantibioticus SPRT was confirmed, suggesting that the PA pathway is present.
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
64
2.2 INTRODUCTION
2.2.1 BIOSYNTHESIS OF DPO
DPO is best known for its properties as a scintillator and as a high efficiency luminophore
(Ionescu et al., 2005; Semenova et al., 2004). It is produced for industry by chemical synthesis,
whereby it is used extensively in laser dyes and as a light activator for liquids (Semenova et
al., 2004; Adrova et al., 1957). However, the biological synthesis of DPO has never been
reported before and therefore its production by S. polyantibioticus SPRT is of great interest.
A biochemical pathway for the production of DPO by S. polyantibioticus SPRT has been
hypothesized whereby the compound is synthesized from starter units of benzoic acid (Figure
2.1A) and phenylalanine or 3-hydroxyphenylalanine (Figure 2.1B). Nucleophilic attack by the
α-amino group of 3-hydroxyphenylalanine on the electrophilic carboxyl group of benzoic acid
would result in the formation of an amide bond and the reaction intermediate benzoyl-β-
hydroxyphenylalanine (Figure 2.1C). This molecule is postulated to undergo a
heterocyclization or cyclodehydration reaction to create an oxazoline intermediate, which
would subsequently be oxidized to form an oxazole, 4-carboxy 2,5-diphenyloxazole (Figure
2.1D), by the nucleophilic attack of the phenylalanine β-hydroxy group on the carbonyl group
of the amide. The decarboxylation of the oxazole moiety would result in the formation of DPO
as the final product (Figure 2.1E).
The formation of the amide bond and subsequent heterocyclization reaction are assumed to be
catalyzed by an NRPS. The NRPS responsible for the biosynthesis of DPO is proposed to have
an A domain, plus ArCP, Cy, Ox, PCP and TE domains (Figure 2.2). The A domain would
recognise either 3-hydroxyphenylalanine or phenylalanine as the substrate and tether it to the
PCP domain. Although it is unclear whether the A domain would bind phenylalanine or β-
hydroxyphenylalanine, it seems more likely that phenylalanine would be the substrate, with
subsequent hydroxylation forming 3-hydroxyphenylalanine. If this is correct, then the DPO
NRPS would be expected to have an additional domain for a P450 monooxygenase to allow
for the β-hydroxylation of phenylalanine before the heterocyclization reaction (this P450
monooxygenase domain is not shown in Figure 2.2). The hydroxylation activity could also be
provided in trans. The ArCP domain would be responsible for loading benzoic acid.
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
65
The PCP domain would be responsible for keeping the 3-hydroxyphenylalanine intermediate
bound to the enzymatic machinery, while the ArCP domain would perform the identical
function in keeping the benzoyl intermediate bound. The Cy domain is proposed to perform
the dual function of facilitating the condensation between benzoic acid and 3-
hydroxyphenylalanine and carrying out the heterocyclization of benzoyl-β-
hydroxyphenylalanine to 4-carboxy 2,5-diphenyloxazole.
Furthermore, the Ox domain, which may exist as an external tailoring enzyme, would be
necessary for the oxidation of the hydrolytically labile oxazoline to form the oxazole moiety in
the heterocyclization reaction (the oxidation of the oxazolidine intermediate is not shown in
Figure 2.1). A decarboxylase reaction would be required to convert 4-carboxy 2,5-
diphenyloxazole to DPO. This decarboxylating activity could also be supplied in trans, as
occurs in curacin A biosynthesis in L. majuscula (Gu et al., 2006). Finally, the TE domain
would be involved in the release of DPO by breaking the covalent linkage between DPO and
the 4'-phosphopantetheine (4'-PP) thiol arm.
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
66
Figure 2.1 Proposed reaction scheme for the synthesis of DPO in S. polyantibioticus SPRT. (A) Benzoic
Acid, (B) 3-Hydroxyphenylalanine, (C) Benzoyl-β-Hydroxyphenylalanine, (D) 4-Carboxy 2,5-
Diphenyloxazole, (E) DPO. Reactions are shown by red arrows or lines, 1- Amide bond
formation, 2- Heterocyclization, 3- Decarboxylation. An NRPS is proposed to catalyse the
condensation of (A) and (B), as well as the proposed heterocyclization of (C) to form (D)
(Stegmann, 2011). Note that the oxazoline intermediate formed by the heterocyclization
reaction is not shown.
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
67
Figure 2.2 The postulated NRPS module arrangement involved in the biosynthesis of DPO. The ArCP and
PCP domains are activated by the transfer of 4’-PP to the conserved serine residue on each
domain, which allows the covalent binding of benzoate (A) and 3-hydroxyphenylalanine (B),
as activated thioester derivatives. Domains: ArCP - aryl carrier protein, Cy – heterocyclization
domain, Ox – oxidation domain, PCP - peptidyl carrier protein domain, TE – thioesterase
domain. (Stegmann, 2011).
2.2.2 ISOLATION OF GENES INVOLVED IN DPO BIOSYNTHESIS
To assess whether the proposed biosynthetic pathway is correct, the genes required for the
synthesis of benzoic acid and the DPO NRPS, which are expected to be clustered on the S.
polyantibioticus SPRT genome, had to be identified.
The identification of an A domain in the genome of S. polyantibioticus SPRT, with a binding
pocket substrate specificity for phenylalanine, would indicate an NRPS that might be
responsible for the biosynthesis of DPO. PCR amplification, using suitable degenerate primers,
would allow for the detection of phenylalanine-specific A domains in S. polyantibioticus SPRT.
The substrate specificities of these PCR-amplified A domains could be determined by
comparison with the binding-pocket specificities of A domains for which the amino-acid
substrates are known. Larger fragments of the DPO biosynthetic gene cluster could then be
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
68
detected by Southern hybridization and further sequencing would reveal the remainder of the
genes constituting the cluster. Identification of the DPO biosynthetic gene cluster would add
to the handful of oxazole biosynthetic gene clusters characterised from actinomycetes
(Pulsawat et al., 2007; Zhao et al., 2006; Onaka et al., 2005).
The identification of a Cy domain in S. polyantibioticus SPRT may be analysed in a similar
fashion to the A domains and their presence would indicate NRPS enzymes involved in making
oxazoles or thiazoles. PCR primers specific for oxazole- and thiazole-producing Cy domains
may be used to amplify such domains.
In addition to searching for NRPS genes, the genes for benzoic acid biosynthesis need to be
identified. The presence of an orthologue of the encP gene, coding for phenylalanine ammonia-
lyase (PAL), in the genome of S. polyantibioticus SPRT would be of great interest, as PAL is a
key enzyme in the generation of benzoyl coenzyme A (benzoyl CoA) via trans-cinnamic acid
in a plant-like manner from phenylalanine in the biosynthesis of enterocin in ‘Streptomyces
maritimus‘strain DSM 41777T (Figure 2.3) (Moore et al., 2002; Hertweck & Moore, 2000). In
spite of its wide occurrence in fungi and plants, benzoic acid is an extremely rare bacterial
metabolite that has only been described in a few bacterial biosynthetic systems and as a central
intermediate of anaerobic aromatic molecule metabolism (Xiang & Moore, 2003). The
existence of an encP orthologue in the genome of S. polyantibioticus SPRT could be elucidated
using suitable PCR primers. Such an encP orthologue would be a strong candidate for
involvement in the biosynthesis of DPO.
If benzoic acid is not synthesized via a PAL-mediated pathway in S. polyantibioticus SPRT, it
could be synthesized via a novel variation on the phenylacetate pathway for the degradation of
phenylalanine. Besides the aerobic process catalysed by PAL in ‘S. maritimus’ strain DSM
41777T, other aerobic and anaerobic pathways for the production of benzoyl-CoA and
derivatives do exist, such as in the β-proteobacterium Azoarcus evansii, in which phenylacetic
acid is degraded via an anaerobic mechanism to benzoyl-CoA (Gescher et al., 2005). It has
been reported that phenylacetate-coenzyme A ligase (PA-CoA ligase) catalyses the initial
reaction in this pathway, which involves the activation of PA to PA-CoA. The gene coding for
PA-CoA ligase, paaK, has also been identified in Streptomyces species (Pometto and Crawford,
1985). Identification of a similar gene to paaK in S. polyantibioticus SPRT could indicate its
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
69
ability to synthesise benzoyl-CoA aerobically from PA (Figure 2.3B). Amino acid catabolism
has been linked to antibiotic synthesis in the production of macrolides in Streptomyces
ambofaciens and Steptomyces fradiae and therefore it is possible that an aromatic amino acid
degradation pathway could be involved in the production of a benzoic acid intermediate for
DPO biosynthesis (Tang et al., 1994). Alternatively, since the chorismate pathway is a common
pathway for generating molecules with benzene rings, such as shikimic acid, in bacteria (and
other organisms), S. polyantibioticus SPRT could perhaps also use a novel variant of one of the
aromatic biosynthetic pathways to generate benzoic acid (Figure 2.3D).
Figure 2.3 Biosynthetic pathways involved in the synthesis of benzoyl-CoA in ‘S. maritimus’ and plants
(route A), in anaerobic bacteria (route B), in fungi (route C) and from shikimic acid (route D)
(Hertweck & Moore, 2000).
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
70
2.3 MATERIALS AND METHODS
2.3.1 STRAINS, MEDIA AND GROWTH CONDITIONS
‘S. maritimus’ DSM 41777T, Streptomyces virginiae NRRL B-1446T, Streptomyces avermitilis
MA-4680T, Streptomyces coelicolor A3(2) and Streptosporangium oxazolinicum JCM 17388T
were grown in yeast extract-malt extract broth (YEME; International Streptomyces Project
medium 2) (Shirling & Gottlieb, 1966) and S. polyantibioticus SPRT was grown in
Streptosporangium Medium (SM) (Pfefferle et al., 2000) for 7 days at 30oC with shaking. Gram
stains and single-colony streaking onto YEME or SM plates were performed to check for
contamination.
2.3.2 GENOMIC DNA EXTRACTION
Total genomic DNA (gDNA) was extracted from bacterial cell mass using the gDNA extraction
method of Wang et al. (1996), with the following modifications: 25 mg lysozyme/ml was used
instead of 5 mg/ml and the cells were incubated in the lysozyme buffer at 37 oC overnight
instead of for 30 min; isoamyl alcohol was omitted from the chloroform extraction step; and
the final precipitation of DNA was performed with the addition of one tenth of a volume of 3
M sodium acetate (pH 5.2) and one volume of room-temperature isopropanol. The
concentration of each gDNA sample was quantitated spectrophotometrically using the
Nanodrop® ND-1000 Spectrophotometer (Coleman Technologies Inc., USA) and analysed by
agarose gel electrophoresis. Visualization was performed with a Gel Doc XR (Bio-Rad
Laboratories Inc., USA) system and Quantity One Version 4.5.2 Software in order to assess
the quantity and integrity of the DNA samples. To confirm that the extracted gDNA was of
good quality, the 16S rRNA gene was PCR amplified as detailed in Cook & Meyers (2003).
gDNA samples were stored at 4 °C.
2.3.3 PRIMER DESIGN
All of the degenerate primers used in the early attempts to isolate the NRPS gene cluster are
listed in Table 2.1, accompanied by their sequences and binding positions. The A3F and A7R
primer set was designed to bind to the conserved motifs of actinomycete A domains, namely
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
71
A3 and A7, respectively (Ayuso-Sacido & Genilloud, 2005). This primer set was used to
amplify NRPS A domain fragments of approximately 700 bp in size.
A multiple amino acid sequence alignment of the Cy-domain regions of one oxazole and four
thiazole producing NRPSs from various bacterial strains (Table 2.2) obtained from the
GenBank database of the National Centre for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov/) was performed using DNAMAN version 4.13 (Lynnon
Biosoft, USA) in order to design the degenerate primer set CyF and CyR. The Cy domain
primer set amplifies a product of 1050 bp from S. polyantibioticus SPRT.
Another two Cy-domain forward primers, VVFTS CycF and QTPQV CycF, were designed
based on a multiple sequence alignment of the amino acid sequences used in the initial primer
design (Table 2.2) plus three new Cy domain sequences from S. coelicolor strain A3(2)
(GenBank accession number: NP_631722), Streptomyces roseosporus strain NRRL 111379
(GenBank accession number: ZP_04712035) and Streptomyces griseus subsp. griseus strain
NBRC 11350 (GenBank accession number: WP_012379765). These forward primers were
used in conjunction with the A domain reverse primer, A7R, and were expected to amplify a
PCR product of 1449 bp.
The primer set, PALHAL_F and PALHAL_R, was designed using DNAMAN from the
consensus sequence of a multiple sequence alignment of the PAL amino acid sequence from
‘S. maritimus’ DSM 41777T (GenBank accession number: AF254925) and the histidine
ammonia lyase (HAL) amino acid sequences from eleven Streptomyces species obtained from
the GenBank database (http://www.ncbi.nlm.nih.gov/) (Table 2.3). An amplification product
of approximately 350 bp was expected to be amplified from S. polyantibioticus SPRT. The
EncP-F and EncP-R primer set was designed to amplify a 721 bp encP fragment from the
biosynthetic gene cluster for benzoyl-CoA-derived enterocin in ‘S. maritimus’ DSM 41777T
(AF254925) (Stegmann, 2011).
The genes involved in the aerobic PA pathway are organized differently in different
Streptomyces strains and therefore two different sets of primers for the amplification of paaK
were designed. The primer set, Paak_AveF and Paak_AveR, was designed based on the
multiple sequence alignment of paaK nucleotide sequences from eight different Streptomyces
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
72
strains obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/), using DNAMAN
(Table 2.4). An amplification product of 490 bp was expected to be amplified from S.
polyantibioticus SPRT. In the same manner, the primer set, Paak_CoeF and Paak_CoeR was
designed based on the multiple sequence alignment of paaK nucleotide sequences from six
different Streptomyces strains using DNAMAN (Table 2.5). An amplification product of 709
bp was expected to be amplified from S. polyantibioticus SPRT.
Table 2.1. Oligonucleotide primers used in this study Primer Name Binding Position Primer Sequence (5'→3') A3F 1120-1142 a GCSTACSYSATSTACACSTCSGG
A7R 1820-1801 a SASGTCVCCSGTSCGGTAS
Cy-F 35-60b AGCCITTCYCSCTSACSSMBSTSCAG
Cy-R 1077-1054c AGGCAGGTCGGAGGTGAAGACGAC
VVFTS CycF 1274-1293d TSGTSTTCACSWSSIHSYTG
QTPQV CycF 1380-1398d TSWSSCAGACSCCSCAGGT
PALHAL_F 571-593e TACGGIGTSWVCACCRGBWTSGG
PALHAL_R 879-902e GIRCASCGBABSGARTASGCGTCC
Paak_AveF 559-583f CCBTCSTACMTGCTSACSCTSCTSGACG
Paak_AveR 1049-1022f GSAGSASGATCTCCTCGARCTGSSTGG
Paak_CoeF 315-340g CCRCGCAGGATSAYCATGTCGTCGC
Paak_CoeR 1024-1001g GCCTCCAGCGGNACSACSGGBCG
EncP-F 766-787e GACTCGCACCTGGCGGTCAAC
EncP-R 1486-1465e GTAGTCGGTGATGGTCTCGTC
M13F 2949-2972 CGCCAGGGTTTTCCCAGTCACGAC
M13R 197-176 TCACACAGGAAACAGCTATGAC aBased on the Brevibacillus brevis grsA1 sequence (GenBank accession number: D00519). bBased on the S. virginiae NRRL B-1446T virH sequence (Table 2.2). c Based on the S. verticillatus strain ATCC 15003 blmIV sequence (Table 2.2). d Based on the S. coelicolor A3(2) non-ribosomal peptide synthetase sequence (GenBank accession number: NP631722) e Based on the ‘S. maritimus’ DSM 41777T encP gene (Table 2.2) f Based on the S. avermitilis MA-4680T paaK gene (Table 2.2)
g Based on the S. coelicolor A3(2) paaK gene (Table 2.2)
Primers use standard ambiguity codes for nucleotides; I = inosine.
‘F’ denotes a forward primer and ‘R’ denotes a reverse primer.
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
73
Table 2.2. Bacterial species and genes used to design the CyF/CyR primers Species name Accession Number Gene Aromatic Compound
Streptoalloteichus hindustanus ATCC 31158 EF032505 tlmIV Tallysomycin Streptomyces atroolivaceus (no strain number given) AF484556 lnmI Leinamycin Streptomyces flavoviridis ATCC 21892 EU670723 zbmIV Zorbamycin Streptomyces verticillatus strain ATCC 15003 AF210249 blmIV Bleomycin Streptomyces virginiae AB283030 virH Virginiamycin M1
Table 2.3. Bacterial species and genes used to design the PALHAL primers Species name Accession Number Protein
‘Streptomyces maritimus’ AF254925 PAL Streptomyces sp. SirexAA-E AEN12031.1 HAL Streptomyces bingchenggensis BCW-1 ADI07714.1 HAL Streptomyces violaceusniger Tü 4113 AEM87830.1 HAL Streptomyces avermitilis MA-4680T WP_037644951.1 HAL
‘Streptomyces cattleya’ NRRL 8057 CCB76521.1 HAL
Streptomyces coelicolor A3(2) NP_629085.1 HAL
Streptomyces pratensis ATCC 33331 ADW03754.1 HAL
Streptomyces fradiae NRRL 18158 KDS89354.1 HAL
Streptomyces griseus NBRC 13350 BAG19440.1 HAL
Streptomyces scabiei 87.22 CBG72492.1 HAL
Streptomyces tsukubaensis NRRL 18488 EIF92105.1 HAL
Table 2.4. Bacterial species and genes used to design the Paak_Ave primers
Species name Accession Number Gene Streptomyces avermitilis MA-4680T BA000030 paaK ‘Streptomyces lividans’ TK24 ACEY01000018 paaK Streptomyces ambofaciens ATCC 23877 AM238663 paaK Streptomyces viridochromogenes DSM 40736 GG657757 paaK Streptomyces bingchenggensis BCW-1 CP002047 paaK Streptomyces albus J1074 DS999645, paaK Streptomyces clavuligerus ATCC 27064 CM000913 paaK Streptomyces sviceus ATCC 27064 CM000951 paaK
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
74
Table 2.5. Bacterial species and genes used to design the Paak_Coe primers
Species name Accession Number Gene Steptomyces coelicolor A3(2) AL645882.2 paaK Streptomyces hygroscopicus ATCC 53653 GG657754 paaK Streptomyces pratensis ATCC 33331 CP002475.1 paaK Streptomyces pristinaespiralis ATCC 25486 CM000950 paaK Streptomyces ghanaensis ATCC 14672 DS999641 paaK Streptomyces sp. SPB78 GG657742 paaK
2.3.4 PCR PROTOCOLS
All PCR amplifications were performed using a Techne TC512 Thermal Cycler fitted with a
heated lid and gradient sample block.
2.3.4.1 A DOMAIN AMPLIFICATION
The cycling conditions used for the amplification of the A domains from S. polyantibioticus
SPRT and Sts. oxazolinicum gDNA using the A3F/A7R primer set was as follows: initial
denaturation at 95 oC for 5 min, followed by 35 cycles of denaturation at 95 oC for 30 s,
annealing at 64 oC for 90 s and elongation at 72 oC for 4 min, with a final elongation at 72 oC
for 10 min. PCR reactions consisted of: 500 ng of DNA, 2 U SuperTherm Taq polymerase
(JMR Holdings, USA), 1.5 μM of each primer, 0.2 mM of each dNTP, 4 mM MgCl2 and 3 %
(v/v) glycerol in a total volume of 50 μl.
2.3.4.2 CY DOMAIN AMPLIFICATION
Cycling conditions for the amplification of the Cy domain from S. polyantibioticus SPRT
gDNA using the CyF/CyR primer set were similar to those used for amplification of the A
domain fragment (section 2.3.4.1), with the exception of the annealing temperature, which was
performed as a gradient ranging from 52 oC to 65 oC. PCR reactions were set up as described
in section 2.3.4.1, with the exception of the MgCl2 concentration, which ranged from 2 mM to
4 mM, the primer concentration, which ranged from 0.5 μM to 2.5 μM for each primer and the
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glycerol concentration, which ranged from 2 % to 8 % (v/v). Template concentration was also
varied from 500 ng to 1500 ng. S. coelicolor A3(2) gDNA was used as a positive control.
Amplification using the VVFTS CycF/A7R and QTPQV CycF/A7R primer sets was performed
using the same cycling conditions and PCR reaction set up mentioned above.
2.3.4.3 AMMONIA-LYASE GENE AMPLIFICATION
Cycling conditions for the amplification of encP from S. polyantibioticus SPRT gDNA, using
the EncP-F/EncP-R primer set, consisted of the following: initial denaturation at 95 oC for 2
min, followed by 30 cycles of denaturation at 95 oC for 45 s, annealing at 60 oC for 30 s and
elongation at 72 oC for 70 s, with a final elongation at 72 oC for 5 min. PCR reactions contained:
500 ng of DNA, 1 U SuperTherm Taq polymerase (JMR Holdings, USA), 0.5 μM of each
primer, 0.2 mM of each dNTP and 4 mM MgCl2, in a total volume of 50 μl. ‘S. maritimus’
DSM 41777T gDNA was used as a positive control for the amplification of the encP fragment.
Cycling conditions using the primer set PALHALF/PALHALR were as follows: initial
denaturation at 95 oC for 5 minutes, followed by 35 cycles of denaturation at 95 oC for 30
seconds, annealing at 56 oC for 30 seconds and elongation at 72 oC for 60 seconds, with a final
elongation at 72 oC for 5 minutes. PCR reactions consisted of: 200 ng of DNA, 2 U SuperThem
Taq polymerase (JMR Holdings, USA), 0.5 μM of each primer, 0.2 mM of each dNTP and 3
mM MgCl2 in a total volume of 20 μl.
2.3.4.4 paaK GENE AMPLIFICATION
Cycling conditions for the amplification of paaK using the primer sets Paak_AveF/Paak_AveR
and Paak_CoeF/Paak_CoeR, from S. polyantibioticus SPRT were as follows: initial
denaturation at 95 oC for 5 min, followed by 35 cycles of denaturation at 95 oC for 30 s,
annealing at 60 oC for 45 s and elongation at 72 oC for 60 s, with a final elongation at 72 oC for
5 min. PCR reactions consisted of: 400 ng of DNA, 2 U SuperThem Taq polymerase (JMR
Holdings, USA), 0.5 μM of each primer, 0.2 mM of each dNTP and 2 mM MgCl2 in a total
volume of 50 μl. S. coelicolor A3(2) and S. avermitilis MA-4680T gDNA were used as a
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76
positive control for the amplification of the paaK fragment using the Paak_CoeF/Paak_CoeR
and Paak_AveF/Paal_AveR primer sets, respectively.
2.3.4.5 LONG RANGE PCR
In order to efficiently amplify large gDNA fragments of 5-25 kb in size, the Expand Long
Range dNTPack (Roche Diagnostics, Germany) was used. The cycling conditions for the
amplification of the DPO gene cluster using the A3F/Paak_AveR, Paak_AveF/A7R, VVFTS
CycF/A7R and QTPQV CycF/A7R primer sets were performed according to the
manufacturer’s instructions. Each PCR reaction consisted of 500 ng gDNA, 0.5 mM of each
dNTP, 0.5 µM of each primer, 8 % (v/v) DMSO, 2.5 mM MgCl2 and 3.5 U Expand Long Range
Enzyme mix in a total volume of 50 µl.
2.3.4.6 COLONY PCR
A colony PCR protocol was used to confirm the presence of cloned inserts in E. coli
transformants harbouring recombinant constructs. The PCR cycling conditions for the
amplification of these inserts was as follows: initial denaturation at 95 oC for 5 min, followed
by 35 cycles of denaturation at 95 oC for 30 s, annealing at 60 oC for 90 s and elongation at 72
oC for 60 s, with a final elongation at 72 oC for 10 min. PCR reactions consisted of: toothpick-
tip size amount of cell mass from an E. coli transformant colony, 2 U SuperThem Taq
polymerase (JMR Holdings, USA), 0.5 μM of each primer, M13F and M13R (Table 2.1), 0.8
mM of each dNTP and 3 mM MgCl2 in a total volume of 20 μl.
All PCR amplification products, including probes used for Southern hybridizations, were
resolved by electrophoresis alongside a λ-PstI molecular marker on 0.8 % agarose gels
containing 0.8 μg/ml ethidium bromide in order to analyse amplicon size and assess primer
specificity. The products were visualized using a long wavelength UV light box (Bio-Rad Gel
Doc EQ-system™, Bio-Rad Laboratories Inc., USA). For cloning, the fragments of interest
were excised from the gel and purified using the FavorPrep Gel/PCR Purification kit
(FavorGenTM, Germany) according to the manufacturer’s instructions. PCR products for
sequencing were purified using the MSB® Spin PCRapace kit (STRATEC Molecular,
Germany).
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2.3.5 SPLINKERETTE METHOD
The splinkerette method, originally described by Devon et al. (1995), was used in an attempt
to obtain additional sequence information both upstream and downstream of the putative
phenylalanine-specific A domain sequence identified by amplification with the A3F/A7R
primer set. The protocol was modified by using an oligonucleotide adaptor sequence that
recognizes a XhoI overhang and XhoI-digested gDNA in the ligation reaction. A standard three
step PCR reaction using an annealing temperature of 55 oC was performed, as described in the
original method, using the splk0/PheAd GSP3 primer set. A second round of PCR was
performed using the same cycling conditions and PCR reagent concentrations, but the
splk1/PheAd GSP2 primer set was used instead and 10 µl of the initial PCR reaction was used
as the template. The primers are listed in Table 2.6.
The PCR amplification products were resolved by electrophoresis alongside a λ-PstI molecular
marker on a 1 % agarose gel, containing 0.8 μg/ml ethidium bromide, in order to analyse
amplicon size and primer specificity. The products were visualized with a long wavelength
UV light box (Bio-Rad Gel Doc EQ-system™, Bio-Rad Laboratories Inc., USA). The
fragments of interest were purified for sequencing as described in section 2.3.4.6.
Table 2.6 Oligonucleotide primers used in the splinkerette method
Oligonucleotide
name Primer Sequence (5'→3') Reference
Splnktop cgaatcgtaaccgttcgtacgagaattcgtacgagaatcgctgtcctctccaacgagccaaga Devon et al. (1995)
Splk0 Cgaatcgtaaccgttcgtacgagaa Devon et al. (1995)
Splk1 tcgtacgagaatcgctgtcctctcc Devon et al. (1995)
PheAd GSP2 tgaacgaggcgaactggagcacc This study
PheAd GSP3 ctgcggcgggatcgtcacatgg This study
Splnkxho tcgagcttggctcgtttttttttgcaaaaa This study
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2.3.6 CLONING AND SEQUENCING
The amplification products obtained from the primer sets Paak_AveF/A7R (2.3.4.4) and
splk1/PheAD GSP2 (section 2.3.5) were purified using the MSB® Spin PCRapace kit
(STRATEC Molecular, Germany) and sent directly for sequencing, whereas the amplification
products obtained from the primer sets A3F/A7R, VVFTS/A7R, Paak_AveF/Paak_AveR,
PALHAL_F/PALHAL_R were ligated individually into the pGEM-T plasmid as described in
the pGEM®-T Easy Vector System kit (Promega, USA). The ligation reaction was incubated
at 22 °C for 14 h after which 10 ng of the reaction was transformed into E. coli α-Select Bronze
Efficiency Competent Cells (Bioline, UK) according to the manufacturer’s instructions. The
transformants were inoculated onto Luria-Bertani agar (LA) (Sambrook et al., 1989)
supplemented with 100 µg/ml ampicillin, 40 µg/ml X-gal and 0.2 mM IPTG and incubated at
37 °C for 18 h. Transformants harbouring recombinant pGEM-T constructs were identified by
blue/white selection and white colonies were subcultured onto fresh LA plates supplemented
with 100 µg/ ml ampicillin, 40 µg/ml X-gal and 0.2 mM IPTG and incubated at 37 °C for 18 h
to allow confirmation of the white phenotype. The presence of the desired inserts was
determined by performing colony PCR (section 2.3.4.6) using the M13F and M13R primer set
(Table 2.1).
Transformants identified as harbouring the correct recombinant constructs were inoculated into
5 ml Luria-Bertani broth (LB) containing 100 µg/ml ampicillin and grown overnight with
shaking at 37 °C. Thereafter, plasmid DNA was isolated using the NucleoSpin Plasmid
Isolation kit (Machery Nagel, Germany) according to the manufacturer’s instructions and
quantitated spectrophotometrically as described before (section 2.3.2). The cloned DNA
fragments were sequenced in both the forward and reverse direction using the M13F and M13R
primers (Table 2.1), by the dideoxy chain-termination method on an Applied Biosystems Big
Dye terminator v3.1 DNA sequencer using BIOLINE Half Dye Mix (Sanger et al., 1977)
(Macrogen Inc., South Korea).
S. polyantibioticus SPRT gDNA, digested with various restriction endonucleases (section
2.3.8.1), was excised from agarose gels and purified using the Favorgen Gel/PCR Purification
Kit (FavorgenTM, Germany), before ligating into the blue/white selection vector, pSK. One
microgram of pSK vector was digested individually with NotI, PstI or SacII overnight at 37 oC
using 1.5 U of each restriction endonuclease along with the appropriate restriction buffer. The
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digested pSK vector was dephosphorylated using rAPid Alkaline Phosphatase (Roche,
Switzerland) according to the manufacturer’s instructions, but the incubation period at 37 oC
was extended to 24 h instead of the recommended 1 h. Subsequently, ligation of the digested
gDNA into the dephosphorylated pSK vector was performed according to the sticky-end
protocol for the Rapid DNA Ligation Kit (Thermo Fisher Scientific, USA) for 14 h at 4 oC,
after which 10 ng of the reaction was transformed into chemically competent E. coli cells.
Transformants were screened for inserts and sequenced as described above.
2.3.7 SEQUENCE ANALYSIS
All sequence chromatograms were viewed and edited using Chromas v2.01, (Technelysium,
Australia). DNA alignments, assembly of nucleotide sequences obtained by PCR
amplification, translations, in silico digestions and other analyses were performed using
DNAMAN v4.13 (Lynnon Biosoft, USA.).
The isolated DNA sequences were used to search for similar sequences in the GenBank
database (http://www.ncbi.blast.nlm.nih.gov/BLAST/) using the BLASTX algorithm (Altschul
et al., 1997). The NRPSpredictor2 program (Rausch et al., 2005) available online (http://www-
ab.informatik.uni-tuebingen.de/software/NRPSpredictor), was used to determine the signature
sequences of the binding pockets of the cloned A domains. The nucleotide sequence of each A
domain was translated and provided to the program, which identified the key binding-pocket
residues by an alignment with the PheA domain of GrsA and determined the probable
specificity of the A domain based on the biochemical and biophysical properties of the binding-
pocket residues.
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2.3.8 SOUTHERN HYBRIDIZATION
2.3.8.1 RESTRICTION ENDONUCLEASE DIGESTION
For Southern hybridizations using the paaK probe, S. polyantibioticus SPRT gDNA was
digested with the restriction endonucleases NotI & PstI and PstI & SacII, as well as with the
single restriction endonucleases NotI, PstI and SacII.
For Southern hybridization using the A domain probe, S. polyantibioticus SPRT gDNA was
digested with the single restriction endonucleases NotI, PstI and SacII.
For Southern hybridization using the encP probe, gDNA of S. polyantibioticus SPRT was
digested with the following restriction endonucleases: SphI & StuI, PvuII & SphI, AvrII & SphI
and AvrII & StuI. ‘S. maritimus’ DSM 41777T genomic DNA was digested with SphI & StuI.
Each reaction consisted of 50 μg of gDNA, 1.5 U of each restriction endonuclease and the
appropriate restriction buffer in a total volume of 50 µl. Digestions were performed overnight
at 37 oC.
2.3.8.2 PROBE PREPARATION
The recombinant constructs harbouring the phenylalanine-specific A domain insert (pGEMA-
18) and paaK gene insert (pGEM-PAAK) served as the templates for probe preparation using
the PCR DIG Probe Synthesis Kit (Roche). Additionally, PCR-amplified encP from ‘S.
maritimus’ DSM 41777T served as a template for probe preparation and was also used in a
Southern hybridization.
Probes for both the A-18 A domain and paaK gene were synthesized using the A domain
(section 2.3.4.1) and paaK (section 2.3.4.4) PCR protocols, respectively, and the plasmid
specific primers, M13F and M13R. The amplified encP gene probe was synthesized using the
DIG Probe Synthesis Kit (Roche, Switzerland), the encP PCR protocol (section 2.3.4.3) and
the EncP-F/EncP-R primer set.
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2.3.8.3 SOUTHERN BLOT HYBRIDIZATION
Restriction endonuclease digested S. polyantibioticus SPRT and ‘S. maritimus’ DSM 41777T
gDNAs were electrophoresed on 0.7 % agarose gels containing 0.8 μg/ml ethidium bromide
and individually blotted onto Hybond N+ membranes (Amersham Biosciences, UK) using a
Trans-blot® SD Semi-dry Transfer Cell (Bio-Rad Laboratories Inc., USA) for 1 h at 15 V and
a current of 355 mA.
Each membrane was air dried and placed into a sealable plastic bag with DIG Easy
Hybridization Buffer (Roche, Switzerland) (approximately 10 ml/100 cm2 of membrane). This
was followed by incubation with gentle shaking for 2 h at 56.3 oC for the A domain probe,
53.8 oC for the encP probe and 48 oC for the paaK probe (these being the calculated optimum
hybridization temperatures for each probe). The hybridization solution was prepared by adding
60 μl denatured probe to 20 ml hybridization buffer (Roche, Switzerland) and hybridization
was performed at the hybridization temperature overnight with gentle shaking. The
temperature of hybridization was calculated using the following formula:
Thyb = TM – (20oC to 25oC)
Where, TM = 49.82 + 0.41(% GC) – 600/ℓ TM – melting point of probe-target hybrid % GC – percentage G+C residues in the probe sequence Thyb – optimal temp for hybridization of the probe to target in DIG Easy Hybridization ℓ – length of the probe/hybrid in base pairs
After hybridization, the membrane was washed twice in low stringency buffer, containing 2 x
standard saline citrate (SSC; 0.3 M NaCl, 30 mM Na3C6H5O7) and 0.1 % sodium dodecyl
sulphate (SDS), at room temperature for 5 min with gentle shaking, followed by washing twice
in high stringency buffer (0.1 x SSC; 0.1 % SDS) at 68 oC for 15 min with gentle shaking. This
was followed by washing with washing buffer for 2 min at room temperature (0.5 M maleic
acid, pH 7.5; 0.3 % Tween 20) and subsequent blocking with 2 % skim milk for 2 h at room
temperature.
Following the blocking step, the membrane was incubated with the anti-DIG Alkaline
Phosphatase conjugate (Roche, Switzerland) at room temperature with shaking for 30 min. The
membrane was washed twice with washing buffer for 15 min at room temperature with shaking
to remove excess antibody.
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82
Before detection, the membrane was equilibrated for 3 min in detection buffer (0.1 M Tris-
HCl, 0.1 M NaCl, pH 9.5) at room temperature. Detection was achieved by incubating the
membrane in 20 ml detection buffer containing 0.175 mg/ml 5-bromo-4-chloro-3-indolyl-
phosphate (BCIP) and 0.25 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) in the dark at room temperature for 0.5-12 h. The reaction was stopped by
rinsing the membrane with Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6) for 5
min, after which the result was digitally captured.
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2.4 RESULTS AND DISCUSSION
2.4.1 ISOLATION OF THE NRPS GENE CLUSTER
2.4.1.1 A DOMAIN AMPLIFICATION FROM S. POLYANTIBIOTICUS SPRT
The identification of an A domain in the genome of strain SPR with a binding pocket substrate
specificity for phenylalanine or 3-hydroxyphenylalanine could indicate that the A domain is
part of the NRPS responsible for the biosynthesis of DPO.
NRPS A domains were amplified from S. polyantibioticus SPRT by primers specific for A
domain conserved motifs (Ayuso-Sacido & Genilloud, 2005) (Figure 2.4). The amplified
products were individually cloned into the pGEM-T Easy vector, transformed into competent
E. coli cells and screened to confirm the presence of the correct size insert using colony PCR.
The inserts were sequenced before being subjected to protein BLAST (BLASTP) analysis in
order to confirm that they were A domains. Thirty-one recombinant constructs were identified
as carrying an insert (ranging in size from 688 to 729 nucleotide bases) with homology to
known A domains in the GenBank database (Table 2.7).
The first 9 amino acid residues of the binding-specificity code of the A domains were identified
for each unique sequence by aligning the protein sequence of each A domain against that of
the PheA domain of GrsA, in a similar manner to that shown in Figure 1.17. This resulted in
the identification of twelve unique amino acid binding pocket codes. Additionally, the A
domain protein sequences were inserted individually into the NRPSpredictor2 program, which
performs an alignment against the PheA domain of GrsA, but also analyses the physico-
chemical fingerprint of the residues lining the amino acid binding pocket to give an indication
of the most probable amino acid specificity of the binding pocket. Due to the occurrence of
relaxed substrate specificity in certain A domains, more than one amino acid substrate can be
predicted for any given A domain (e.g. substrates with similar physico-chemical properties)
(Rausch et al., 2005). The specificity binding code and likely amino acid substrate specificity
for the twelve unique A domains obtained from S. polyantibioticus SPRT are listed in Table
2.7.
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84
Figure 2.4 Gel electrophoresis of A domain amplicons from S. polyantibioticus SPRT amplified by PCR
using the degenerate primer set A3F/A7R. Lanes: 1- λ-PstI molecular marker, 2-6 – different
A domains amplified from S. polyantibioticus SPRT gDNA.
The sequences of the inserts in clones pGEMA-18 and pGEMA-66 were identical and were
determined by the NRPSpredictor2 to be specific for phenylalanine. Protein BLAST analysis
of the amino acid sequence of both inserts showed a high similarity to an A domain in
Granuliella mallensis MP5ACIX8 (GenBank accession number: YP_00507340.1) with an
amino acid similarity of 57 %, an A domain in Streptomyces griseus XYLEBKG1 (GenBank
accession number: YP_08236938.1) with an amino acid similarity of 60 % and an A domain
in Streptomyces netropsis (GenBank accession number: BAH_68437.1) with an amino acid
similarity of 54 %. Additionally, all of these BLASTP hits had an amino acid substrate
specificity for phenylalanine, as predicted by the NRPSpredictor2 program.
When comparing the amino acid specificity code of the pGEMA-18/pGEMA-66 clones to the
well-known phenylalanine-specific A domains of GrsA and BarG, there are differences
observed at the 236, 239, 299, 330 and 331 positions (Table 2.7). At position 236, pGEMA-
11.54
5.08 2.84 1.70
0.81 0.55
kb 1 2 3 4 5 6
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85
18/A-66 differs from GrsA and BarG in having a cysteine residue instead of an alanine residue.
However, according to the gross clustering of NRPS substrates by chemical similarity, both
alanine and cysteine are classified as aliphatic, hydrophobic residues and thus could perform
the same function in the binding pocket interacting with non-polar, hydrophobic phenylalanine
(Rausch et al., 2005). At position 299, pGEMA-18/A-66 displays an alanine residue instead of
isoleucine in the GrsA sequence and valine in the BarG sequence. Isoleucine, alanine and
valine are, however, all non-polar amino acids, which may make them interchangeable, and
therefore they could perform the same function in the binding pocket in interacting with
phenylalanine. The same argument can also be used to explain the differences at position 330,
where GrsA displays an isoleucine residue in comparison to valine in both GrsA and pGEMA-
18/A-66.
The key differences are at position 239 where GrsA and BarG both display a tryptophan residue
in comparison to a glycine residue in pGEMA-18/A-66 and at position 331 where GrsA and
BarG both present a cysteine residue in contrast to aspartic acid in pGEMA-18/A-66.
However, both of these key differences occur at positions of medium to high variability and
thus should not have a large impact on the substrate affinity, as these residues may have
minimal interaction with the substrate amino acid.
From the specificity-conferring code and NRPS prediction software, it is possible to infer that
the pGEMA-18/pGEMA-66 A domain may be involved in the recognition and activation of
phenylalanine as a starter molecule in the biosynthesis of DPO. However, it is also possible
that the pGEMA-18/pGEMA-66 A domain may have a substrate specificity for amino acids
other than phenylalanine and this is due to the fact that the NRPSpredictor2 predicts aromatic
substrates less reliably due to the observed promiscuity of the A domains utilizing these
substrates (Rausch et al., 2005).
Furthermore, as serine residues are common amino acids for the biosynthesis of oxazoles, it is
feasible that any of the A domains specific for serine, such as the inserts in clones pGEMA-5,
A-7, A-12, A-15, A-22, A-36, A-51 and A-63 may activate serine as the starter unit instead of
phenylalanine, thereby utilising it in the biosynthesis of DPO (Roy et al., 1999). However, if
serine is recognised by the A domain involved in DPO biosynthesis, then it would require the
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86
addition of a phenyl group to carbon-3 of the serine residue via an unusual β-phenylation
reaction.
The identification of the genes surrounding the phenylalanine A domain, as well as the A
domains specific for serine, would help to elucidate the DPO biosynthetic strategy. However,
attempts at isolating additional sequence information upstream and downstream of the A
domain specific for phenylalanine using the splinkerette method failed, as non-specific product
amplification was observed (data not shown).
Table 2.7 Specificity Binding Pocket Code and Amino Acid Specificity
Adenylation domain Source
Specificity Binding Pocket Code Residue and Positiona Amino Acid Specificityb
235 236 239 278 299 301 322 330 331 517
GrsA D A W T I A A I C K Phe, Trp, Phg
BarGc D A W T V A A V C K Phe, Trp, Phg
Clone pGEMAD-2 D V Q F N A H M V - Pro
Clone pGEMAD-16 D A F F L G V T F - Ile, Leu, Val
Clone pGEMA-1 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-5 D F W N V G M V H - Ser, Thr, Dht
Clone pGEMA-7 D F W N V G M V H - Ser, Thr, Dht
Clone pGEMA-8 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-11 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-12 D F W N V G M V H - Ser, Thr, Dht
Clone pGEMA-14 D M V Q F G L V H - Gly, Ala, Val
Clone pGEMA-15 D V W H F S L I D - Ser, Thr
Clone pGEMA-16 D A F F L G A T F - Ile, Leu, Val
Clone pGEMA-18 D C G T A A A V D - Phe, Trp, Phg, Tyr
Bht
Clone pGEMA-19 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-22 D F W N V G M V H - Ser, Thr, Dht
Clone pGEMA-28 D M E N L G L I N - Orn, Lys, Arg
Clone pGEMA-31 D A F F L G A T F - Ile, Leu, Val
Clone pGEMA-32 G I Y H L G L L C - Dhpg, hpg
Clone pGEMA-33 G I Y H L G L L C - Dhpg, hpg
Clone pGEMA-36 D V W H F S L I D - Ser, Thr
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87
Clone pGEMA-40 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-48 - - - D V G F V - - NO HIT
Clone pGEMA-51 D V W H F S L I D - Ser, Thr
Clone pGEMA-53 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-58 D A F F L G V T F - Val, Leu, Ile
Clone pGEMA-60 D A F F L G A A T - Val, Leu, Ile
Clone pGEMA-63 D V W H F S L I D Ser, Thr
Clone pGEMA-66 D C G T A A A V D - Phe, Trp, Phg, Tyr,
Bht
Clone pGEMA-68 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-69 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-70 D M V Q F G L V Y - Gly, Ala, Val
Clone pGEMA-74 D A A D V G F V D - Glu, Gln, Asp, Asn
Variabilityd % 3 16 16 39 52 13 26 23 26 0
a According to GrsA numbering b Predictions in order of decreasing preference, where A domain specificities have been clustered according to the physico-chemical fingerprint of the residues lining the amino acid binding pocket to give an indication of the most probable amino acid specificity of the binding pocket. c Part of the barbamide gene cluster in Lyngbya majuscule (GenBank accession number: AAN32981), a cyanobacterium. d The amount of variation observed at each amino acid position in the A domain substrate-determining signature sequence. The most variable positions (278 and 299) are considered to be ‘wobble’ positions (similar to the third position in DNA codons (Stachelhaus et al., 1999). Residue position 517 was not obtained for S. polyantibioticus SPRT A domain clones but, based on the high conservation of this residue, it is most likely lysine. Amino acid abbreviations use standard one and three letter codes. Dht - dehydrothreonine, Phg - L-phenylglycine, Hpg – hydroxyphenylglycine, Dhpg – dihydroxyphenylglycine. Highlighted rows indicate the clones carrying an A domain insert specific for phenylalanine.
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2.4.1.2 SOUTHERN HYBRIDIZATION USING THE PHENYLALANINE-
SPECIFIC A DOMAIN PROBE
A Southern hybridization experiment was performed using a collection of single restriction
endonuclease digestions (PstI, NotI and SacII) of S. polyantibioticus SPRT gDNA in an attempt
to isolate DNA fragments large enough to provide additional sequence data on the NRPS gene
cluster involved in DPO biosynthesis. The phenylalanine-specific A domain probe detected
numerous bands of varying size, ranging from 0.7 kb to 4.5 kb, for each restriction
endonuclease in the Southern hybridization experiment (Figure 2.5). The presence of multiple
DNA fragments generated by the SacII and NotI digestions may be explained as a result of the
target A domain sequence being cleaved by these enzymes. An in silico digest of the S.
polyantibioticus SPRT phenylalanine (Phe)-specific A domain probe revealed that it is digested
by SacII at position 49 and 577 and by NotI at position 518. However, PstI does not cleave
within the Phe-specific A domain and thus it is proposed that the multiple hybridization bands
generated during this digestion may indicate the presence of other Phe-specific A domains in
the genome.
Due to the fact that the sizes of these bands were known, gel electrophoresis of gDNA digested
with PstI, SacII and NotI was performed and DNA of the appropriate size was purified and
cloned. Unfortunately, sequencing of the inserts did not yield any A-domain sequences, other
NRPS domains or their flanking regions.
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Figure 2.5 Southern hybridization of restriction endonuclease digested S. polyantibioticus SPRT gDNA
using the Phe-specific A domain as a probe. Lanes 1-4 each contain 50 µg of S. polyantibioticus
SPRT gDNA digested singly with the following enzymes; Lanes: 1- PstI, 2- SacII 3- NotI, 4-
Unlabelled Probe (positive control).
2.4.1.3 CY DOMAIN AMPLIFICATION
Initially, the degenerate Cy domain PCR primer set, CyF/CyR, was designed based on
sequences from characterised Streptomyces and Streptoalloteichus thiazole and oxazole
producers and was expected to amplify a fragment of approximately 1050 bp from S.
polyantibioticus SPRT gDNA. However, there was no amplification of the correct-sized band
from either S. polyantibioticus SPRT gDNA or S. virginiae NRRL B-1446T (Figure 2.6A). It
should be noted that S. virginiae NRRL B-1446T may not be the same strain as that used for
1 2 3 4
4.5 2.9 0.7
kb
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the design of the CyF/CyR primers (the papers describing virginiamycin M biosynthesis do not
specify which S. virginiae strain was used). As it is not known whether strain NRRL B-1446T
has the biosynthetic genes for virginiamycin M1, it may not have been a suitable positive
control.
Consequently, two new degenerate Cy domain PCR forward primers, VVFTS CycF and
QTPQV CycF, were designed based on a more comprehensive multiple sequence alignment
consisting of the original Cy domain sequences and the addition of three new Streptomyces Cy
domains. The new primers were used in conjunction with the A7R primer and were expected
to amplify a product of approximately 1449 bp. The reason for using a Cy-domain forward
primer with an A-domain reverse primer is that a typical linear NRPS elongation module
consists of the core domains arranged in the canonical order of C/Cy-A-PCP. An amplification
product of the correct size was observed using the VVFTS CycF/A7R primer set, but
sequencing revealed that a non-target product had been amplified. This was explained as being
due to the single VVFTS CycF primer on its own amplifying non-target sequences (Figure
2.6B, lane 9). The primer set QTPQV CycF/A7R failed to amplify the correct size product
(Figure 2.6C).
The lack of amplification of a Cy domain fragment may have been due to the fact that the target
sequence is not present in the genome of S. polyantibioticus SPRT, but a more likely scenario
is that the primers did not bind to the target sequences in S. polyantibioticus SPRT. A lack of
primer binding is plausible given the low degree of homology observed in the multiple
sequence alignments of Cy domains from the various thiazole and oxazole producers. The lack
of amplification in the case of the VVFTS CycF/A7R and QTPQV CycF/A7R primer sets may
be due to the fact that the NRPS biosynthetic strategy for DPO production may not follow the
conventional linear NRPS arrangement, but may in fact consist of a non-linear organization or
even contain unusual freestanding A or C/Cy domains. Investigation of this hypothesis is
described in the following chapter on the S. polyantibioticus SPRT genome sequence.
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
91
Figure 2.6 Gel electrophoresis of PCR amplified DNA from S. polyantibioticus SPRT using the degenerate
Cy domain primer sets (A) CyF/CyR, (B) VVFTS CycF/A7R and (C) QTPQV CycF/A7R. (A)
Lane 1: λ-PstI molecular marker, Lane 2-4: Amplification at annealing temperature of 52 oC
and increasing MgCl2 concentrations of 2, 3 and 4 mM respectively, Lane 5-7: Amplification at
annealing temperature of 58 oC and increasing MgCl2 concentrations of 2, 3 and 4 mM
respectively, Lane 8-10: Amplification at annealing temperature of 65 oC and increasing MgCl2
concentrations of 2, 3 and 4 mM respectively, Lane 11: No template control, Lane 12:
Amplification of Streptomyces virginiae NRRL B-1446T gDNA at annealing temperature of 52 oC and MgCl2 concentration of 3 mM . (B) Lane 1: λ-PstI molecular marker, Lane 2-4:
Amplification at annealing temperature of 55 oC and increasing MgCl2 concentrations of 2, 3
and 4 mM, respectively, Lane 5-7: Amplification of S. coelicolor (A3)2 gDNA at annealing
temperature of 55 oC and increasing MgCl2 concentrations of 2, 3 and 4 mM respectively, Lane
8: No template control, Lane 9: VVFTS CycF primer only, Lane 10: A7R primer only. (C) Lane
1: λ-PstI molecular marker, Lane 2: No template control, Lane 3-5: Amplification at annealing
temperature of 55 oC and increasing MgCl2 concentrations of 2, 3 and 4 mM respectively, Lanes
6-8: Amplification of S. coelicolor (A3)2 gDNA at annealing temperature of 55 oC and
increasing MgCl2 concentrations of 2, 3 and 4 mM respectively, Lane 9: QTPQV CycF primer
only.
11.54
5.08 2.84 2.56 1.99 1.70 1.16 0.81
0.51
A 1 2 3 4 5 6 7 8 9 10 11 12
kb 11.54 5.08 1.70 1.16
11.54 5.08
1.70 1.16
kb
kb
B
C
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9
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2.4.1.4 NRPS DOMAIN AMPLIFICATION FROM STS. OXAZOLINICUM
Sts. oxazolinicum was recently characterised as producing a novel group of antibiotics, the
spoxazomicins, which contain an oxazole moiety and are therefore likely to be produced non-
ribosomally (Inahashi et al., 2011). In light of this, it was decided that amplification of an A
domain and/or Cy domain from Sts. oxazolinicum would aid in the identification of the correct
A domain for DPO biosynthesis in S. polyantibioticus SPRT.
Subsequently, two unique A domains, bearing substrate specificity for serine and glycine
respectively, were identified in Sts. oxazolinicum following their amplification using the
A3F/A7R primer set. The Cy domain amplification using the VVFTS CycF/A7R and QTPQV
CycF/A7R primer sets was unsuccessful. Due to the fact that Cy domains are absolutely
necessary for the heterocyclization of serine or cysteine in the formation of an oxazole or
thiazole heterocyclic ring, respectively, it was decided that the A domains in S. polyantibioticus
SPRT that are specific for serine would remain of significant interest in the quest to uncover
the biosynthetic pathway involved in DPO production. Investigation into this hypothesis is
described in the following chapters.
2.4.2 ISOLATION OF GENES INVOLVED IN BENZOIC ACID BIOSYNTHESIS
2.4.2.1 AMPLIFICATION OF PAL/HAL
It has been shown that the presence of the PAL-encoding gene, encP, is absolutely required for
benzoyl-CoA formation in ‘S. maritimus’ DSM 41777T and therefore encP is a prime candidate
to screen for when searching for benzoyl-CoA biosynthetic potential (Xiang & Moore, 2003).
PCR amplification was performed using the designed encP primers on gDNA extracted from
S. polyantibioticus SPRT and ‘S. maritimus’ DSM 41777T. Amplification was observed for ‘S.
maritimus’ DSM 41777T gDNA resulting in a clear, single band of about 0.7 kb (Figure 2.7).
There was no similar amplification observed for encP amplification from S. polyantibioticus
SPRT. The lack of amplification from S. polyantibioticus SPRT DNA could be because the
organism does not contain an encP homologue. However, it could also be because the design
of the EncP-F/EncP-R primers was based on a single sequence (from ‘S. maritimus’ DSM
41777T), containing no variable nucleotide positions and therefore may not have bound
efficiently to the S. polyantibioticus SPRT encP homologue.
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The PAL amino acid sequence from ‘S. maritimus’ DSM 41777T was used in a multiple
sequence alignment together with the HAL amino acid sequences from 11 Streptomyces strains
in order to design new degenerate primers for the amplification of a PAL/HAL from S.
polyantibioticus SPRT. The HAL sequences from various Streptomyces strains share a high
degree of homology with each other and to the PAL sequence of ‘S. maritimus’ DSM 41777T.
A 350 bp fragment was amplified from S. polyantibioticus SPRT gDNA, which was sequenced
and identified as a HAL after analysis using BLASTX. It was concluded that the existence of
a PAL in the genome of S. polyantibioticus SPRT is unlikely and therefore that the synthesis of
benzoic acid for incorporation into DPO does not proceed via a PAL-catalysed conversion of
phenylalanine to trans-cinnamic acid. Benzoic acid could be produced in a novel manner in S.
polyantibioticus SPRT, perhaps via the phenylalanine degradation pathway mentioned earlier
(section 2.2.2).
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
94
kb 11.54 5.08 2.84 2.56 1.99 1.70 1.16 0.81
Figure 2.7 Gel electrophoresis of PCR amplified encP and 16S rRNA genes from S. polyantibioticus SPRT
and ‘S. maritimus’ DSM 41777T. Lanes: 1- λ-PstI molecular marker, 2- ‘S. maritimus’ DSM
41777T amplified 16S rRNA gene, 3- S. polyantibioticus SPRT amplified 16S rRNA gene, 4-
‘S. maritimus’ DSM 41777T amplified encP, 5-6 S. polyantibioticus SPRT with no amplified
encP.
2.4.2.2 SOUTHERN HYBRIDIZATION USING THE ENCP GENE PROBE
Southern hybridization was performed using various pairwise restriction endonuclease
digestions of S. polyantibioticus SPRT gDNA along with a pairwise restriction endonuclease
digestion of ‘S. maritimus’ DSM 41777T gDNA. There was no hybridization observed for any
of the S. polyantibioticus SPRT genomic digests, while a weak hybridization band was observed
for the ‘S. maritimus’ DSM 41777T positive control (data not shown). The fact that no
hybridization signal was detected, corroborates the result of the PCR amplification experiment
(section 2.4.2.1) and suggests that there is no encP homologue in S. polyantibioticus SPRT.
However, neither the PCR nor the Southern hybridization result rules out the possibility that
there is an encP homologue in S. polyantibioticus SPRT involved in the synthesis of benzoic
1 2 3 4 5 6
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95
acid, but which has a very dissimilar nucleotide sequence to the ‘S. maritimus’ DSM 41777T
encP gene.
2.4.2.3 AMPLIFICATION OF pAAK
Despite S. polyantibioticus SPRT appearing not to possess a PAL-like gene similar to encP, it
may produce benzoyl-CoA via the phenylacetate pathway used for the degradation of
phenylalanine. The initial step of the phenylacetate pathway is catalyzed by a PA-CoA ligase
and degenerate primers, paaK_AveF and paak_AveR, were used to amplify a 490 bp fragment
of the homologue of the gene, paaK, encoding this enzyme in S. polyantibioticus SPRT (Figure
2.8). The degenerate primer set, paaK_CoeF and paaK_CoeR did not amplify the expected
size product from S. polyantibioticus SPRT gDNA (data not shown). Additionally, based on
the fact that genes encoding functions for the biosynthesis of many antibiotics are clustered,
the primer sets A3F/paak_AveR and paak_AveF/A7R, were used in an effort to amplify larger
gDNA fragments containing both an NRPS A domain and the phenylacetate pathway gene
cluster (Ikeda et al., 1999). These attempts proved unsuccessful (data not shown).
The presence of paaK within the genome of S. polyantibioticus SPRT suggests that the
phenylacetate pathway is present, which was expected, as the prototrophic S. polyantibioticus
SPRT should have all the pathways for amino acid biosynthesis and degradation. However,
whether a derivative of this pathway is used by S. polyantibioticus SPRT to produce benzoyl-
CoA remains to be determined. It is possible that the genes involved in benzoic acid synthesis
are clustered together with the NRPS involved in DPO biosynthesis and therefore sequencing
further upstream and downstream of the amplified paaK fragment may help to uncover the
DPO NRPS.
Chapter 2 – Early attempts at isolating the gene cluster responsible for DPO biosynthesis in S. polyantibioticus SPRT
96
Figure 2.8 Gel electrophoresis of PCR amplified paaK from S. polyantibioticus SPRT and S. avermitilis
MA-4680T. Lanes: 1- λ-PstI molecular marker, 2 - S. polyantibioticus SPRT amplified paaK
using 2 mM MgCl2 in PCR reaction, 3 - S. polyantibioticus SPRT amplified paaK using 3 mM
MgCl2 in PCR reaction, 4 - S. polyantibioticus SPRT amplified paaK using 4 mM MgCl2 in
PCR reaction 5- S. avermitilis MA-4680T amplified paaK.
2.4.2.4 SOUTHERN HYBRIDIZATION USING THE PAAK GENE PROBE
Southern hybridization using the paaK probe resulted in the detection of single bands of
approximately 1.8 kb for S. polyantibioticus SPRT gDNA digested with SacII, 2.5 kb for gDNA
digested with NotI and 4.5 kb for gDNA digested with PstI (Figure 2.9). S. polyantibioticus
SPRT digested gDNA fragments of about 1.8 kb and 2.5 kb were gel purified and cloned into
the plasmid vector pSK, before using colony PCR to identify clones carrying inserts.
Sequencing of the 1.8 kb insert revealed the presence of a monooxygenase gene and sequencing
of the 2.5 kb insert identified an A domain specific for proline. No clones containing the paaK
gene were identified. Thus, these results did not establish that the monooxygenase gene and
the proline A domain are near the paaK gene, nor that the paaK gene is near an NRPS
containing an A domain that activates phenylalanine.
1 2 3 4 5 kb 11.54
5.08 2.84 1.70
1.16 0.81 0.50
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Figure 2.9 Southern hybridization of restriction endonuclease digested S. polyantibioticus SPRT genomic
DNA using the amplified paaK gene as a probe. Lanes 1-6 contain single and double
endonuclease digestions of 50 µg of S. polyantibioticus SPRT genomic DNA in each Southern
hybridization. Lanes: 1- NotI/PstI, 2- NotI, 3- PstI, 4- PstI/SacII, 5– SacII, 6- Unlabelled Probe
(positive control).
1 2 3 4 5 6 kb 4.5
2.5 1.8 0.5
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98
2.5 CONCLUSION
In the efforts to isolate and identify the biosynthetic gene cluster for the production of DPO in
S. polyantibioticus SPRT, twelve unique A domains were identified. Importantly, one A
domain was predicted to be specific for the activation of phenylalanine, while two A domains
were predicted to be specific for serine. Serine could be involved in DPO biosynthesis due to
its known involvement in heterocyclic ring formation.
This chapter describes the extensive efforts to isolate additional sequence information upstream
and downstream of the NRPS A domains of interest, using the techniques of Southern
hybridization, the splinkerette method and long-range PCR. These efforts were all
unsuccessful. Furthermore, the inability to identify an NRPS Cy domain or any genes involved
in benzoic acid biosynthesis, led to the conclusion that a completely different approach was
required, namely, sequencing of the full S. polyantibioticus SPRT genome. The sequencing of
the genome and its annotation are described in the following chapter.
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2.6 REFERENCE LIST
Adrova, N. A., Koton, M. M., & Florinsky, F. S. (1957). Preparation of 2, 5-diphenyloxazole and its scintillation efficiency in plastics. Russian Chemical Bulletin, 6(3): 394-395. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25: 3389-3402. Ayuso-Sacido, G. & Genilloud, O. (2005). New PCR Primers for the sequencing of NRPS and PKS-I Systems in Actinomycetes: Detection and Distribution of These Biosynthetic Gene Sequences in Major Taxonomic Groups. Microbial Ecology, 49: 10-24. Cook A.E. & Meyers P.R. (2003). Rapid identification of filamentous actinomycetes to the genus level using genus-specific 16S rRNA gene restriction fragment patterns. International Journal of Systematic Evolutionary Microbiology, 6: 1907-1915. Devon, R.S., Porteous, D.J. & Brookes, A.J. (1995). Splinkerettes--improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Research, 23(9): 1644–1645. Gescher, J., Eisenreich, W., Worth, J., Bacher, A. & Fuchs, G. (2005). Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme. Molecular Microbiology, 56(6): 1586-1600. Gu, L., Jia, J., Liu, H., Håkansson, K., Gerwick, W.H. & Sherman, D.H. (2006). Metabolic coupling of dehydration and decarboxylation in the curacin A pathway: functional identification of a mechanistically diverse enzyme pair. Journal of the American Chemical Society, 128: 9014-9015. Hertweck, C. & Moore, B.S. (2000). A plant-like biosynthesis of Benzoyl-CoA in the marine bacterium ‘Streptomyces maritimus’. Tetrahedron, 56: 9115-9120. Ikeda, H., Nonomiya, T., Usami, M., Ohta, T. & Mura, S.O. (1999). Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proceedings of the National Academy of Sciences of the USA, 96: 9509-9514. Inahashi, Y., Iwatsuki M., Ishiyama A., Namatame M., Nishihara-Tsukashima A., Matsumoto A., Hirose T., Sunazuka T., Yamada H., Otoguro K., Takahashi Y., Omura S. & Shiomi K. (2011). Spoxazomicins A-C, novel antitrypanosomal alkaloids produced by an endophytic actinomycete, Streptosporangium oxazolinicum K07-0460(T). The Journal of Antibiotics (Tokyo), 64(4): 303-307. Ionescu, S., Popovici, D., Balaban, A.T. & Hillebrand, M. (2005). Experimental and theoretical study of 2,5-diaryloxazoles whose aryl are para-substituted phenyl groups. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 62(1-3): 252-60.
Moore, B.S., Hertweck, C., Hopke, J.N., Izumikawa, M., Kalaitzis, J.A., Nilsen, G., O'Hare, T., Piel, J., Shipley, P.R., Xiang, L., Austin, M.B. & Noel, J.P. (2002). Plant-like biosynthetic pathways in bacteria: from benzoic acid to chalcone. Journal of Natural Products, 65: 1956-1962.
Onaka, H., Nakaho, M., Hayashi, K., Igarashi, Y. & Furumai, T. (2005). Cloning and characterization of the goadsporin biosynthetic gene cluster from Streptomyces sp. TP-A0584. Microbiology, 151: 3923-3933.
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Pfefferle, C., Theobald, U., Gürtler, H. & Fiedler, H. (2000). Improved secondary metabolite production in the genus Streptosporangium by optimization of the fermentation conditions. Journal of Biotechnology, 80: 125-142. Pometto III, A.L., & Crawford, D.L. (1985). L-Phenylalanine and L-tyrosine catabolism by selected Streptomyces species. Applied Environmental Microbiology, 49(3): 727-729. Pulsawat, N., Kitani, S. & Nihira, T. (2007). Characterization of biosynthetic gene cluster for the production of virginiamycin M, a streptogramin type A antibiotic, in Streptomyces virginiae. Gene, 393: 31-42. Rausch, C. Weber, T., Kohlbacher, O., Wohlleben, W. & Huson, D.H. (2005). Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs). Nucleic Acids Research, 33: 5799-808.
Roy, R.S., Gehring, A.M., Milne, J.C., Belshaw, P.J. & Walsh, C.T. (1999). Thiazole and oxazole peptides: biosynthesis and molecular machinery. Natural Product Reports, 16: 249 – 263.
Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989). Bacterial media, antibiotics and bacterial strains. Molecular Cloning, a laboratory manual, 2nd Ed. Cold Spring Harbour: Cold Spring Harbour Laboratory Press. Sanger, F., Nicklen, S. & Coulson, A., R. (1977). DNA Sequencing with chain-terminating inhibitors. Biotechnology, 24: 104-108. Semenova, O.N., Galkina, O.S., Patsenker, L.D., Yermolenko, I.G. & Fedyunyayeva, I.A. (2004). Experimental and theoretical investigation of the reaction of 2,5-diphenyl-1,3-oxazole and 2,5-diphenyl-1,3,4-oxadiazole dimethylamino derivatives with the Vilsmeier reagent. Functional Materials, 11: 67–75. Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology, 16: 313-340. Stachelhaus, T., Mootz, H.D, & Marahiel, M.A. (1999). The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chemistry & Biology, 6: 493-505. Stegmann, D.E. (2011). The Investigation into the synthesis of 2, 5-diphenyloxazole in Streptomyces polyantibioticus SPRT. M.Sc. Thesis, Department of Molecular and Cell Biology, University of Cape Town. Tang, L., Zhang, Y. & Hutchinson, C.R. (1994). Amino Acid Catabolism and Antibiotic Synthesis: Valine Is a Source of Precursors for Macrolide Biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae. Journal of Bacteriology, 176(19): 6107-6119. Wang, Y., Zhang, Z. & Ruan, J. (1996) A proposal to transfer Microbispora bispora (Lechevalier, 1965) to a new genus, Thermobispora gen. nov., as Thermobispora bispora combo. nov. International Journal of Systematic Bacteriology, 46: 933 – 938.
Xiang, L. & Moore, B.S. (2003). Characterization of Benzoyl Coenzyme A Biosynthesis Genes in the Enterocin-Producing Bacterium “Streptomyces maritimus”. Journal of Bacteriology, 185: 399-404.
Zhao, C., Ju, J., Christenson, S. D., Smith, W. C., Song, D., Zhou, X. Shen, B. & Deng, Z. (2006). Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning the oxazolomycin biosynthetic gene cluster from Streptomyces albus JA3453. Journal of Bacteriology, 188: 4142–4147.
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CHAPTER 3
STREPTOMYCES POLYANTIBIOTICUS SPRT GENOME
EXPLORATION 3.1 Abstract ...................................................................................................................... 103
3.2 Introduction ................................................................................................................ 105
3.3 Materials and Methods ............................................................................................... 111
3.3.1 Genomic DNA extraction .............................................................................. 111
3.3.2 Ethanol precipitation of genomic DNA ......................................................... 111
3.3.3 Genome sequencing and assembly using the 454 stand-alone platform ........ 111
3.3.3.1 Genome annotation ............................................................................ 111
3.3.4 Genome sequencing and assembly using a hybrid approach ......................... 112
3.3.4.1 Genome annotation ............................................................................ 112
3.3.4.2 Sequence analysis .............................................................................. 112
3.4. Results and Discussion .............................................................................................. 114
3.4.1 Initial genome sequencing using 454 technology .................. 114
3.4.2 S. polyantibioticus SPRT genome properties ......................... 114
3.4.3 Identification of the putative DPO biosynthetic gene cluster 117
3.4.4 Investigation into the putative DPO gene cluster .................. 119
3.4.4.1 A domain specificity .................................................. 124
3.4.4.2 Cy domains ................................................................ 126
3.4.4.3 PCP domains .............................................................. 135
3.4.4.4 Ox domains ................................................................ 139
3.4.4.5 TE domain .................................................................. 140
3.4.4.6 Genes involved in benzoic acid biosynthesis ............. 141
3.5 Conclusion ................................................................................................................. 144
3.6 Reference list ............................................................................................................. 145
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CHAPTER 3
STREPTOMYCES POLYANTIBIOTICUS SPRT GENOME
EXPLORATION
3.1 ABSTRACT
Initial efforts to sequence the S. polyantibioticus SPRT genome using the 454 pyrosequencing
approach proved unsuccessful. However, a hybrid approach using the “third generation” NGS
technology, PacBio, in combination with the Illumina MiSeq approach provided a draft
S. polyantibioticus SPRT genome sequence and with it the framework to identify the putative
DPO biosynthetic cluster. The draft S. polyantibioticus SPRT genome sequence was annotated
using antiSMASH 3.0, which identified 43 secondary metabolite gene clusters, thereby
revealing the potential of this strain to produce a range of biotechnologically pertinent
compounds.
A gene cluster within the draft S. polyantibioticus SPRT genome was identified as the putative
DPO biosynthetic gene cluster due to the fact that the NRPS genes in this cluster more closely
resembled the enzyme architecture hypothesized to be involved in DPO biosynthesis than any
other secondary metabolite cluster identified in the genome. The gene content and the
arrangement of the genes in the proposed DPO gene cluster was identical to the congocidine
biosynthetic gene cluster identified in Streptomyces ambofaciens ATCC 23877T.
Analysis of the NRPS domains within the putative DPO biosynthetic gene cluster revealed a
nonlinear NRPS initiation module, two lone C domains, a lone PCP domain and a lone A
domain encoded by an acyl-CoA synthetase. The in silico prediction of the substrate specificity
of the A domains within both the initiation module and the acyl-CoA synthetase proved
inconclusive due to the different predictions obtained from a range of NRPS prediction
programmes. However, it was hypothesized that both A domains could display a degree of
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104
relaxed amino acid substrate specificity, commonly observed in A domains displaying
specificity for aromatic amino acids, thereby allowing one A domain to activate phenylalanine
and the other to activate benzoic acid, in line with the hypothetical DPO biosynthetic scheme
presented in Chapter 2 (Figure 2.1). Furthermore, a phylogenetic analysis in combination with
an analysis of the conserved signature motifs frequently observed in NRPS domains revealed
the presence of S. polyantibioticus SPRT genes encoding putative Cy and TE domains within
the proposed DPO biosynthetic cluster. In addition, one PCP domain within the putative DPO
biosynthetic cluster was identified as likely to be active from the conserved signature motif
analysis, while a second PCP domain was classified as inactive due to the absence of a key
catalytic residue. Lastly, a biosynthetic pathway for benzoyl-CoA production in S.
polyantibioticus SPRT is proposed, based on the genome analysis.
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3.2 INTRODUCTION
The explosion in microbial genome sequencing over the past decade and a half, which has
appropriately been named the genomics era, has boosted the field of drug discovery from
microbial natural products. The chain-termination DNA sequencing method, also referred to
as dideoxy sequencing, developed by Sanger and colleagues in the 1970s has remained the
most commonly employed DNA sequencing technique to date. Recently, however, this method
has been partially superseded by several next-generation sequencing (NGS) technologies that
offer attractive increases in cost-effective sequence throughput (Morozova & Marra, 2008).
The automated chain-termination (Sanger) method is considered as a ‘first-generation’
technology, and newer methods are referred to as next-generation sequencing. These newer
technologies constitute various strategies that rely on a combination of template preparation,
sequencing and imaging, and genome alignment and assembly methods (Metzker, 2011;
Niedringhaus et al., 2011).
A major limitation of the chain-termination method is the requirement of in vivo PCR
amplification of DNA fragments that need to be sequenced, which is usually accomplished by
the laborious and labour intensive practice of cloning into bacterial hosts (Morozova & Marra,
2008). The 454 sequencing technology (Figure 3.1) developed by Roche Applied Science
(Penzberg, Germany) was the first NGS technology released onto the market and bypasses the
cloning requirement by using a highly efficient in vitro DNA amplification method known as
emulsion PCR (Tawflik & Griffiths, 1998). During emulsion PCR, individual DNA fragments
are linked to specific 454-adaptors bound to single streptavidin-coated beads which are
suspended in a water-in-oil emulsion, so that each bead with a single DNA fragment resides in
an individual aqueous droplet. These discrete emulsion droplets act as distinct amplification
reactors producing approximately 1 million clonal copies of a unique DNA template per bead
(Margulies et al., 2005). Each template-containing bead is then transferred into the well of a
picotiter plate and the templates are analysed using pyrosequencing. Instead of using
dideoxynucleotides to terminate the chain amplification, pyrosequencing technology relies on
the detection of pyrophosphate released during nucleotide incorporation and can be described
as a sequencing-by-synthesis technique that measures the release of inorganic pyrophosphate
(PPi) by chemiluminescence (Liu et al., 2012; Morozova & Marra, 2008). Briefly, the template
DNA is immobilized, and solutions of dNTPs are added sequentially; the release of PPi,
Chapter 3 – Streptomyces polyantibioticus SPRT genome exploration
106
whenever the complementary nucleotide is incorporated, is detectable by light produced by a
chemiluminescent enzyme, such as luciferase, present in the reaction mix. The sequence of the
DNA template is determined from a “pyrogram,” which corresponds to the order of correct
nucleotides incorporated. Since chemiluminescent signal intensity is proportional to the
amount of pyrophosphate released and hence the number of bases incorporated, the
pyrosequencing approach is prone to errors that result from incorrectly estimating the length
of homopolymeric sequence stretches (i.e. indels). The current state-of-the-art 454 platform
marketed by Roche Applied Science (Penzberg, Germany) is capable of generating 80–120 Mb
of sequence in 200- to 300-bp reads in a 4 h run (Morozova & Marra, 2008).
Figure 3.1 The 454 pyrosequencing approach. In this sequencing approach, DNA is isolated, sheared and
ligated to special adaptors. Emulsion PCR is then performed for clonal amplification after which the pyrosequencing reaction takes place using a mixture of the single-stranded DNA template, the sequencing primer, and the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase (Siqueira et al., 2012).
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Other “second generation” NGS technologies include the Illumina MiSeq/Solexa approach,
which involves sequencing-by-synthesis of single-molecule arrays with reversible terminators.
A solid glass surface, known as a flowcell, which consists of an acrylamide coating, is used to
immobilize individual DNA molecules and bridge PCR amplification (PCR amplification that
occurs between primers bound to a surface) is used to amplify DNA into clusters of identical
molecules. This sequencing method is similar to the chain-termination method, in that it also
relies on dye terminator nucleotides incorporated in the sequence by a DNA polymerase.
However, Illumina/Solexa terminators are reversible, permitting polymerization to proceed
even after fluorophore detection. DNA sequencing commences with addition of the sequencing
primer, DNA polymerase, and four reversible dye terminators. Fluorescence is recorded after
incorporation by a four-channel fluorescent scanner (Quail et al., 2012; Siqueira et al., 2012;
Glenn, 2011). Furthermore, massively parallel sequencing by hybridization–ligation, which
was implemented in the supported oligonucleotide ligation and detection system (SOLiD), was
developed by Applied Biosystems (Waltham, USA) (Morozova & Maraa, 2008). This
approach involves fluorescent probes that undergo repetitive steps of hybridization and ligation
to complementary positions in the template strand, followed by fluorescence imaging to
identify the ligated probe (Loman et al., 2012). The ligation chemistry used in SOLiD is based
on the polony sequencing technique that was published in the same year as the 454 method
(Niedringhaus et al., 2011; Shendure et al., 2005). Meanwhile, “third generation” NGS
technologies include the PacBio Single Molecule, Real-Time (SMRT®) DNA Sequencing
System developed by Pacific Biosystems (Menlo Park, USA) that provides the longest read
lengths of any available sequencing technology (Roberts et al., 2013; Niedringhaus et al.,
2011). “Third-generation sequencing” is comprised of two main characteristics; 1) PCR is not
required before sequencing, which reduces DNA preparation time for sequencing and 2) the
signal is captured in real time, which means that the signal, no matter whether it is fluorescent
(Pacbio) or an electric current (Nanopore), is monitored during the enzymatic reaction of
adding a nucleotide to the growing complementary strand (Liu et al., 2012).
SMRT® sequencing is a sequencing-by-synthesis technology based on real-time imaging of
fluorescently tagged nucleotides as they are incorporated along individual DNA template
molecules. Due to the fact that the technology uses a DNA polymerase to drive the reaction,
and because it images single molecules, there is no degradation of signal over time (Roberts et
al., 2013). SMRT® is based on a chip pioneered by Levene et al. (2003) that contains an array
of zero-mode waveguides (ZMWs). Due to the fact that biological systems characteristically
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pack their molecules at spacing much smaller than the wavelength of visible light, fluorescent
microscopy techniques that target single-molecule studies must overcome the diffraction-
limited resolution of conventional optical microscopy. Fluorescence microscopy techniques
such as ZMWs have been developed for this purpose and consist of simple nanostructures that
allow real-time observation of individual molecules at high concentrations (Zhu & Craighead,
2012).
During SMRT® sequencing, a single DNA polymerase is attached to the bottom of a well on a
SMRT® Cell, and the millions of ZMWs create an illuminated volume that is small enough to
observe the incorporation of a single nucleotide. Each time a nucleotide is added to the DNA
at the bottom of the well, the dye is detected before being cleaved off and diffusing away (Liu
et al., 2012; Levene et al., 2003). The camera inside the instrument computer captures the
signal in real-time observation (Mardis, 2013; Liu et al., 2012).
The Ion Torrent platform, marketed by Life Technologies (Carlsbad, USA), uses a sequencing
strategy similar to the 454 platform, except that hydrogen ions (H+) are detected instead of a
pyrophosphatase cascade. The use of H+ means that no lasers, cameras or fluorescent dyes are
needed (Glenn, 2011). Furthermore, this platform utilizes semiconductor technology that
detects the release of protons as nucleotides are incorporated during DNA synthesis. DNA
fragments with specific adapter sequences are linked to and then clonally amplified by
emulsion PCR on the surface of 3-micron diameter beads, known as Ion Sphere Particles. The
templated beads are loaded into proton-sensing wells that are fabricated on a silicon wafer and
sequencing is primed from a specific location in the adapter sequence. As sequencing proceeds,
each of the four bases is introduced sequentially and if bases of that type are incorporated,
protons are released and a signal is detected proportional to the number of bases incorporated
(Quail et al., 2012).
Nanopore sequencing is another “third generation sequencing” method that relies on the transit
of a DNA molecule or its component bases through a biological nanopore. The bases are
detected by their effect on an electrical current or optical signal (Steinbock & Radenovic, 2015;
Schadt et al., 2010).
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The increase in speed and decrease in the cost of genome sequencing, as well as the rise of
metagenomic sequencing projects, have been the key driving factors for the revolution in the
field of microbial drug discovery. Particularly, it has been revealed that streptomycetes still
represent a major source for the discovery of novel natural products due to the wealth of NRPS
and PKS structural diversity that they encode (Boddy, 2014). This was emphasized by the
complete genome sequence of S. coelicolor strain A3(2), which revealed the unexpected
potential of this microorganism to produce natural products that were undetectable by classical
screening methods, such as bioactivity-guided fractionation of crude fermentation broth
extracts and chemical screening methods (Aigle et al., 2014; Bachmann et al., 2014).
Indeed, whereas just five secondary metabolites were identified over 50 years using classical
screening approaches, the advancement in genome sequencing technologies and bioinformatics
has revealed that S. coelicolor A3(2) has the capability to produce almost 20 additional natural
products (Bentley et al., 2002).
Furthermore, the mining of the genome of S. ambofaciens ATCC 23877T which, for many
decades, was only known to produce the macrolide spiramycin and the pyrrolamide
congocidine, has allowed for the identification of an additional 23 gene clusters putatively
involved in the production of a variety of other secondary metabolites.
Importantly, it is critical to note that this situation is not specific to S. coelicolor A3(2) and
S. ambofaciens ATCC 23877T, as both complete and partial genome sequencing of numerous
streptomycetes has shown that they all contain a large number of cryptic secondary metabolite
gene clusters. Thus, these strains contain genes that are not expressed under standard laboratory
conditions or the products are formed at a level too low to be detected in laboratory growth
conditions (Aigle et al., 2014; Ikeda et al., 2014, Jensen et al., 2014, Nett et al., 2009; Baltz,
2008).
The annotation of sequenced genomes from several Streptomyces species has shown that a
single strain can carry more than 30 secondary metabolite gene clusters, thereby supporting the
idea that the biosynthetic potential of this bacterial genus is far from being fully exploited
(Aigle et al., 2014).
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The recent advances in genome sequencing are rapidly changing the field of bacterial natural
products research by providing opportunities to assess the biosynthetic potential of strains prior
to chemical analysis or biological testing. Furthermore, the easy access to sequence data is
driving the development of new bioinformatic tools and methods to identify the products of
silent or cryptic pathways (Jensen et al., 2014; Bérdy, 2012).
Indeed, automatic bioinformatics platforms, such as antiSMASH 3.0 (Weber et al., 2015),
allow the efficient detection of secondary metabolite gene clusters belonging to a range of
different classes of natural products and also facilitate the semi-automated prediction of the
structure of the natural products encoded by these secondary metabolite blueprints (Bachmann
et al., 2014). It has been reported that antiSMASH analysis, which has become the gold
standard for mining secondary metabolite gene clusters in genome sequences, compares
favourably with high-quality, detailed manual annotation of bacterial whole genomes and
provides a more detailed description of individual gene clusters identified than NaPDOS
(Ziemert et al., 2012) and NP.searcher (Li et al., 2009) (Boddy, 2014; Harrison & Studholme,
2014). Furthermore, the ClusterBlast and SubClusterBlast tools available within antiSMASH
enable rapid identification of unique gene clusters from sequence data sets (Boddy, 2014).
However, a concern with automated genome analysis is that gene clusters located near to each
other, may be merged into superclusters, as occurs with the salinilactam gene cluster (Udwary
et al., 2007), as antiSMASH defines clusters as groups of signature genes within 10 kb of each
other and extends the cluster 20 kb on each side of the last signature gene to delineate the
boundaries of PKS and NRPS biosynthetic gene clusters (Boddy, 2014; Medema et al., 2011).
Nevertheless, the recent innovation in genome sequencing has disclosed the astonishing wealth
of new polyketide and non-ribosomal peptide natural product diversity to be mined from
genetic data and these web-based bioinformatics tools can play a key role in guiding the
discovery of novel natural products in genome mining projects, enabling the focus on
biosynthetic gene clusters likely to encode new natural product diversity (Boddy, 2014).
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3.3 MATERIALS AND METHODS
3.3.1 GENOMIC DNA EXTRACTION
The gDNA for both 454 sequencing and sequencing using the hybrid approach was extracted
from S. polyantibioticus SPRT bacterial cell mass using the gDNA extraction method described
in section 2.3.2.
3.3.2 ETHANOL PRECIPITATION OF GENOMIC DNA
Total gDNA isolated from S. polyantibioticus SPRT was subjected to ethanol precipitation so
that it would be stable against any fluctuations in temperature during transportation to ChunLab
(Seoul, South Korea) for hybrid genome sequencing. Briefly, 1 µl of 10 mg/ml glycogen was
added to 100 µl of 1 µg/µl gDNA sample, followed by the addition of 10 µl of 3 M sodium
acetate (pH 5.2), after which the sample was briefly vortexed. Three hundred microliters of
100 % ethanol was then added to the sample, which was vortexed briefly again. The sample
was placed on ice for 30 min and thereafter transported to the sequencing facility.
3.3.3 GENOME SEQUENCING AND ASSEMBLY USING THE 454 STAND-ALONE
PLATFORM
The first attempt at sequencing the S. polyantibioticus SPRT genome was performed at the Next
Generation Sequencing Facility at the University of the Western Cape (Cape Town, South
Africa) using a 454 GS FLX Titanium system (Roche, Switzerland). The Newbler Assembler
2.6 (Roche, Switzerland) was used in an attempt to assemble the reads.
3.3.3.1 GENOME ANNOTATION
Initially, the genes in each of the individual contigs obtained from the 454 genome sequencing
were predicted using the Rapid Annotation using Subsystem Technology (RAST) 2.0 server
database (Overbeek et al., 2014). Additionally, the sequences of the contigs were aligned and
edited using Sequencher version 5.1 (Genecodes Corporation, USA) in order to achieve
assembly.
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3.3.4 GENOME SEQUENCING AND ASSEMBLY USING A HYBRID APPROACH
The genome of S. polyantibioticus SPRT was sequenced at ChunLab (Seoul, South Korea)
using a combination of an Illumina Miseq PE-300 system (Illumina, USA) with 2 × 300 paired-
end reads and a PacBio RSII system (Pacific Biosciences, USA). The Illumina and PacBio
reads were assembled using CLC Genomics Workbench 7.0.4 (CLCbio, Denmark) and PacBio
SMRT Analysis 2.2.0, respectively, and used to construct the draft S. polyantibioticus SPRT
genome sequence.
3.3.4.1 GENOME ANNOTATION
The genes in the assembled genome were predicted using the Rapid Annotation using
Subsystem Technology (RAST) 2.0 server database (Overbeek et al., 2014) and the gene-caller
GLIMMER 3.02 (Delcher et al., 2007). The predicted ORFs were annotated by searching
clusters of orthologous groups (COGs) using the SEED database (Disz et al., 2010; Tatusov et
al., 1997). CLgenomics™ 1.06 (ChunLab) was used to visualize the genomic features, while
protein homologies and conserved domains were analysed by application of BLASTp and the
Conserved Domain Database (CDD; Marchler-Bauer et al., 2011). AntiSMASH 3.0 (Weber
et al., 2015) was used to identify secondary metabolite clusters within the draft genome, while
the NRPSpredictor2 program (Röttig et al., 2011) in combination with the latent semantic
indexing (LSI) model (developed by Baranasic et al., 2013), the NRPS/PKS prediction server
(Bachmann & Ravel, 2009), the Non-Ribosomal Peptide Synthase Substrate Predictor
(NRPSsp) (Preito et al., 2012) and the Stachelhaus code (Stachelhaus et al., 1999) were used
to predict the substrate specificity of all contigs containing NRPS A domains. The NaPDos
tool (available via http://napdos.ucsd.edu/) was utilized in the analysis of all NRPS C domains
(Ziemert et al., 2012). Other sequence features were identified using RNAmmer (Lagesen et
al., 2007) and tRNAscan-SE (Lowe & Eddy, 1997).
3.3.4.2 SEQUENCE ANALYSIS
For the phylogenetic analysis of the putative domains identified as members of the DPO
biosynthetic cluster, amino acid sequences of each separate domain, A, C, PCP, Ox and TE,
were obtained from the detailed antiSMASH 3.0 annotation and aligned against homologous
amino acid sequences that were selected from the GenBank database
(http://www.ncbi.nlm.nih.gov/). The sequences were aligned using the Multiple Sequence
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Comparison by Log-Expectation (MUSCLE) algorithm (Edgar, 2004) in Molecular
Evolutionary Genetics Analysis (MEGA) version 6.0 (Tamura et al., 2013).
Phylogenetic analyses were performed by construction of unrooted phylogenetic trees using
the neighbour joining (Saitou & Nei, 1987), maximum likelihood (Jones et al., 1992) and
maximum parsimony (Takahashi & Nei, 2000) methods. A bootstrap test was employed, based
on 1000 replicates, to assess the reliability of the topology of each phylogenetic tree. All
columns in the alignment containing gaps and missing data were eliminated from the dataset.
Additionally, the conserved NRPS signature motifs, as described by Schwarzer et al. (2003),
were examined in the multiple sequence alignment of the putative S. polyantibioticus SPRT A,
C, PCP, Ox and TE domains in comparison to homologous reference sequences obtained from
the GenBank database (http://www.ncbi.nlm.nih.gov/).
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3.4 RESULTS AND DISCUSSION
3.4.1 INITIAL GENOME SEQUENCING USING 454 TECHNOLOGY
The 454 pyrosequencing platform generated 7 178 853 bp of S. polyantibioticus SPRT genome
sequence consisting of 3553 contigs, however, the de novo assembly of the contigs, ranging in
size from 100 bp to 78 914 bp, proved unsuccessful. This was attributed to the inability of the
sequence assembly program to deal with the highly repetitive strings of G + C residues.
In an attempt to manually assemble the contigs, the sequence analysis program Sequencher
version 5.1 (Genecodes Corporation, USA) was employed, but this strategy was unsuccessful.
After trying to work with the 454 sequence data for over a year, it was concluded that the reason
the genome-sequence contigs could not be assembled was due to there being pieces of the
genome missing. Indeed, S. polyantibioticus SPRT has been characterized to use salicin and
myo-inositol as sole carbon sources (Le Roes-Hill & Meyers, 2009) yet the RAST analysis
showed that critical enzymes, such as the protein-Nπ-phosphohistidine sugar
phosphotransferase (EC 2.7.1.69) involved in the catabolism of salicin to salicin-6-phosphate
and all of the enzymes involved in myo-inositol metabolism were absent. Additionally,
S. polyantibioticus SPRT has been characterized to use L-phenylalanine as a sole nitrogen
source (Le Roes-Hill & Meyers, 2009), yet the deaminating phenylalanine dehydrogenase
(EC 1.4.1.20), critical for its catabolism, was also absent from the sequencing data. The absence
of these key enzymes, in addition to critical enzymes involved in fatty acid metabolism,
menaquinone biosynthesis and glycolysis/gluconeogenesis, prompted the decision to
re-sequence the S. polyantibioticus SPRT genome using a different platform.
3.4.2 S. POLYANTIBIOTICUS SPRT GENOME PROPERTIES
The Illumina platform provided 482.63-fold coverage (for a total of 15 763 940 sequencing
reads) of the genome, while the PacBio platform generated a 38.13-fold coverage of the
genome (for a total of 65 082 sequencing reads). The assembled sequences from the two
approaches resulted in 1 scaffold that consisted of 12 large contigs, while the assembled draft
genome comprised a chromosome (Appendix A) with a length of 8 821 522 bp and 71.53 % G
+ C content (520.76-fold coverage), which is lower than the 74.4 ± 0.2 mol% determined by
the thermal denaturation method (Le Roes-Hill & Meyers, 2009). The draft genome sequence
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contained 8148 ORFs, of which the majority (5360, 66%) were assigned putative functions
according to the COG functional categories (Table 3.1). In addition, the draft genome sequence
consisted of 69 tRNA genes and 24 rRNA genes. Le Roes-Hill and Meyers (2009) reported 7
rRNA operons in the description of Streptomyces polyantibioticus sp. nov. The existence of
69 tRNA genes is somewhat surprising considering the fact that there are 64 possible codons
(of which three are stop codons and would not be involved in the activation of amino acids). It
may be noted that the Streptomyces davawensis JCM 4913 genome sequence also revealed the
presence of 69 tRNA genes, which suggests that the occurrence of more than the usual 30-40
tRNA encoding genes in bacterial cells is not uncommon (Jankowitsch et al., 2012; Lodish et
al., 2000).
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Table 3.1. Number of genes associated with the general COG functional categories Description Value % of totala
Translation, ribosomal structure and biogenesis
215
4.01
Transcription 633 11.81
Replication, recombination and repair 169 3.15
Cell cycle control, cell division, chromosome partitioning 41 0.76
Posttranslational modification, protein turnover, chaperones 147 2.74
Cell wall/membrane/envelope biogenesis 302 5.63
Cell motility 8 0.15
Inorganic ion transport and metabolism 177 3.30
Signal transduction mechanisms 356 6.64
Energy production and conversion 304 5.67
Carbohydrate transport and metabolism 461 8.60
Amino acid transport and metabolism 490 9.14
Nucleotide transport and metabolism 125 2.33
Coenzyme transport and metabolism 226 4.22
Lipid transport and metabolism 321 5.99
Secondary metabolite biosynthesis, transport and catabolism 159 2.97
General function prediction only 766 14.29
Function unknown 460 8.58
a The total is based on the 8148 ORFs identified in the annotated genome.
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3.4.3 IDENTIFICATION OF THE PUTATIVE DPO BIOSYNTHETIC GENE
CLUSTER
AntiSMASH 3.0 identified 43 gene clusters encoding secondary metabolites in the draft
genome of S. polyantibioticus SPRT, revealing the potential of this strain to produce a wide
range of biotechnologically relevant compounds: 4 clusters for lantipeptides, 6 clusters for
NRPSs, 1 cluster for a siderophore, 10 clusters for PKSs, 2 hybrid NRPS-PKS clusters, 6
clusters encoding terpenes, 2 clusters encoding bacteriocins, 2 clusters encoding melanin, 1
cluster encoding a phenazine/melanin hybrid, 1 cluster encoding a β-lactam, 1 cluster encoding
a terpene/lantipeptide hybrid, 1 cluster encoding a lantipeptide/lassopeptide/terpene hybrid, 1
cluster encoding an aminoglycoside/aminocyclitol, 1 cluster encoding a butyrolactone, 1
cluster encoding an ectoine and 3 unidentified clusters.
The putative biosynthetic scheme for the production of DPO proposes that a single NRPS
condenses a molecule of benzoic acid with 3-hydroxyphenylalanine. This NRPS is proposed
to possess a single A domain, plus ArCP, Cy, Ox, PCP and TE domains (Figure 2.2). In light
of this hypothesis, the antiSMASH results were analysed to identify the genes involved in the
biosynthesis of DPO amongst the NRPS gene clusters.
The secondary metabolite gene cluster spanning the region located from nucleotide 637827-
688770 (Appendix A) of the draft S. polyantibioticus SPRT genome consisted of a linear NRPS
encoding 3 A domains with two of them predicted to be specific for the activation of ornithine
and one specific for the activation of threonine. Additionally, all of the genes comprising this
cluster shared homology to the genes comprising the coelichelin biosynthetic cluster identified
in S. coelicolor A3(2).
An NRPS gene cluster spanning the region located from nucleotide 669778-735243 (Appendix
A) overlapped the NRPS cluster mentioned above, but consisted of 2 A-PCP di-domains and
freestanding, adjacently encoded A, C and PCP domains. The three A domains were predicted
to be specific for the activation of threonine, proline or valine and leucine, isoleucine, isovaline
or 2-amino-butyric acid, respectively. The composition of this gene cluster was most similar
in gene content to the tobramycin biosynthetic gene cluster identified in Streptoalloteichus
tenebrarius, as 24 % of the S. polyantibioticus SPRT genes shared homology to genes within
this cluster according to the antiSMASH analysis.
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Due to the fact that both of the NRPS gene clusters mentioned above consisted of 3 A domains
each, of which none were predicted to be specific for the activation of aromatic amino acids
such as phenylalanine or 3-hydroxyphenylalanine, and because the homologous gene clusters
encode coelichelin and tobramycin, neither of which contains an aromatic heterocycle, they
were dismissed as putative DPO biosynthetic clusters.
The secondary metabolite gene cluster spanning the region located from nucleotide 3093585 –
3154158 (Appendix A) consisted of 4 linear NRPS modules in close proximity and also
overlapped an upstream secondary metabolite cluster encoding a terpene, which was predicted
to encode a compound of complex molecular structure. Indeed, the gene content of this gene
cluster shared the highest homology (12 %) with the biosynthetic gene cluster encoding the
cyclic depsipeptide, skyllamycin A, isolated from Streptomyces sp. Acta 2897. The
skyllamycin A structure consists of a cinnamoyl side chain and incorporates a large number of
β-hydroxylated amino acids as well as an unusual α-hydroxyglycine moiety as a rare structural
modification (Pohle et al., 2011). This structure does not bear any resemblance to that of DPO
and this gene cluster was therefore dismissed as a putative DPO biosynthetic cluster.
Similarly, the secondary metabolite cluster spanning the region from nucleotide 3320540 –
3386004 (Appendix A) consisted of 4 linear NRPS modules, in addition to an A-PCP di-
domain, as well as lone A and PCP domains, thus also predicted to encode a molecule of
complex structure. The gene content of this gene cluster shared the highest overall homology
(17 %) to the calcium-dependent cyclic lipopetide antibiotic gene cluster identified in
S. coelicolor (A3)2. Additionally, the composition and arrangement of the genes shared 100
% sub-cluster homology with the clinically important glycopeptide antibiotic teicoplanin
isolated from Actinoplanes teichomyceticus. Due to the structural dissimilarities between DPO
and teicoplanin, this gene cluster was ruled out as the putative DPO biosynthetic cluster.
The secondary metabolite gene cluster spanning the region located from nucleotide 5343994-
5398013 (Appendix A) consisted of 2 A-PCP di-domains, 1 NRPS elongation module, as well
as 2 freestanding C domains and a freestanding PCP domain. The A domains within this gene
cluster were predicted to be specific for the activation of valine, aspartate and alanine, therefore
ruling out the formation of a heterocycle such as DPO.
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The secondary metabolite gene cluster spanning the region from nucleotide 5634984-5665006
(Appendix A) consisted of a nonlinear NRPS module which deviated from the classical C-A-
PCP domain organization and consisted of a lone A domain encoded by an acyl-CoA
synthetase, two lone C domains, a lone PCP domain and a lone TE domain. All of the genes
within this cluster shared homology to the congocidine biosynthetic gene cluster identified in
S. ambofaciens ATCC 23877T. Due to the fact that this cluster was significantly smaller than
any of the other secondary metabolite clusters encoding NRPSs and because the A domains
were predicted to be specific for the activation of aromatic amino acids, it was explored in
greater depth.
3.4.4 INVESTIGATION INTO THE PUTATIVE DPO GENE CLUSTER
The gene cluster spanning the region on the draft S. polyantibioticus SPRT genome from
positions 5634984-5665006 was preliminarily identified as the DPO biosynthetic gene cluster
based on the antiSMASH 3.0 analysis and due to the fact that the NRPS genes comprising this
cluster most closely resembled the gene architecture hypothesized to be involved in DPO
biosynthesis, in comparison to the remainder of the secondary metabolite clusters identified in
the genome. The genes in this cluster were further analysed with BLASTp to determine protein
homologies and conserved domains (Table 3.2).
The in silico analysis identified an atypical NRPS, consisting of a freestanding nonlinear
module and several single-domain proteins encoded by four genes, as the components of the
putative DPO biosynthetic cluster. Interestingly, the composition of the gene cluster and the
arrangement of the genes were most similar to the pyrrole-amide congocidine (also known as
netropsin) (Figure 3.2) biosynthetic gene cluster identified in S. ambofaciens ATCC 23877T,
as 100 % of the S. polyantibioticus SPRT genes shared homology to the genes within the
congocidine cluster. Congocidine biosynthesis is characterised by an NRPS that displays some
unusual features, namely that the A domain acts iteratively and the C domain uses a CoA-
activated substrate rather than a PCP-phosphopantetheinyl-activated substrate. Additionally,
the organization of the only complete NRPS module, A-PCP-C, differs from the canonical C-
A-PCP organization and the remaining NRPS-associated domains are freestanding (Juguet et
al., 2009).
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Figure 3.2 Outline of the chemical structure of congocidine.
Freestanding A and PCP domains, such as the A domain encoded by gene SPR_52860 and the
PCP encoded by SPR_53070, are normally found when a particular amino acid is modified
before its incorporation into the secondary metabolite, such as found in the biosynthesis of the
aminocoumarins and vancomycin (Juguet et al., 2009; Fischbach & Walsh, 2006).
Freestanding C domains, such as those encoded by SPR_52900 and SPR_53040, as well as
those involved in the biosynthesis of vibriobactin, are, however, far less frequent. The
condensation reactions catalysed by freestanding C domains generally necessitate a PCP-
tethered biosynthetic intermediate and a nucleophilic substrate free in solution (Juguet et al.,
2009; Keating et al., 2002), such as the ArCP-bound benzoyl intermediate proposed during
DPO biosynthesis.
There are 24 genes constituting the putative DPO biosynthetic cluster starting with SPR_52860
and ending at SPR_53090 (Figure 3.3). Table 3.2 presents an overview of the predicted
function of each of the products of the genes, as well as their proposed role in DPO
biosynthesis. Two of the genes, SPR_52870 and SPR_52880, are predicted to confer resistance
to DPO and one of the genes, SPR_52890, is predicted to encode a regulator for DPO
biosynthesis. Out of the remaining 21 genes, 5 genes, SPR_52900, SPR_53040, SPR_53060,
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SPR_53070 and SPR_53090, are predicted to encode NRPS-associated domains, while
another, SPR_52860, encodes an enzyme belonging to the AMP-binding superfamily of
proteins and highly resembles acyl-CoA synthetases. The role of the proteins encoded by the
remaining 15 genes cannot be easily inferred from the in silico analysis. Indeed, the presence
of 5 genes, SPR_52960 to SPR_53000, encoding proteins involved in sugar biosynthesis is
perplexing, as DPO does not contain a sugar moiety. It is possible that a glycosylated form of
DPO is synthesised by S. polyantibioticus SPRT, however, this putative glycosylated derivative
has not been isolated. It is also possible that the putative glycosyltransferase encoded by
SPR_52980 and the putative mannanase encoded by SPR_53010 are involved in a self-
resistance mechanism involving the inactivation of DPO by intracellular glycosylation and
extracellular reactivation by hydrolysis of the added sugar, similar to the type of mechanism
demonstrated in Streptomyces antibioticus (Juguet et al., 2009; Quirós et al., 1998).
The genes, cgc4, cgc5, cgc6 and cgc7, present in the congocidine biosynthetic pathway and
homologous to SPR_52920, SPR_52930, SPR_52940 and SPR_52950, respectively, were
characterized as having no involvement in the biosynthesis of congocidine (Lautru et al., 2012).
Similarly, it is possible that these genes play no part in DPO biosynthesis.
Although the putative DPO gene cluster closely resembles both the type and order of the genes
present in the congocidine biosynthetic cluster, there are a number of differences, namely: 1)
the mannanase encoded by SPR_53010 is absent in congocidine biosynthesis, 2) the TE domain
encoded by SPR_53090 is absent in congocidine biosynthesis, and 3) the A domains encoded
by SPR_52860 and SPR_53060 confer specificity towards different substrates than the A
domains involved in congocidine biosynthesis.
In light of this and due to the existence of transposases encoded by SPR_49500 and
SPR_57250, it is possible that S. polyantibioticus SPRT acquired the region between the genes
encoding these enzymes in the form of a transposable genetic element, via horizontal gene
transfer facilitated by the transposases. The G + C content of this region is 72.5 %, which is
slightly higher than the G + C content of the S. polyantibioticus SPRT draft genome (71.53 %),
thereby suggesting that the transposable genetic element may have been acquired from another
actinomycete, such as Streptomyces netropsis DSM 40093 or S. ambofaciens ATCC 23877T.
It also suggests that S. polyantibioticus SPRT has adapted the pathway for pyrrolamide
biosynthesis for the production of DPO.
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Table 3.2 Components of the putative DPO biosynthetic cluster and their proposed
function
Gene ID Size (aa)
GenBank accession number
Closest species match
Identity (%)
Putative function Proposed role in DPO biosynthesis
SPR_52860 518 AHF81557 Streptomyces netropsis DSM
40093
65 Acyl-CoA synthetase DPO assembly
SPR_52870 626 AHF81556 Streptomyces netropsis DSM
40093
75 ABC transporter, ATP-binding protein
Resistance
SPR_52880 616 AHF81555 Streptomyces netropsis DSM
40093
74 ABC transporter, transmembrane
protein
Resistance
SPR_52890 268 AHF81554 Streptomyces netropsis DSM
40093
57 Two-component response regulator
Regulation
SPR_52900 478 AHF81553 Streptomyces netropsis DSM
40093
62 NRPS, C domain Not involved
SPR_52910 479 CAJ88625 Streptomyces ambofaciens ATCC
23877
71 Aldehyde dehydrogenase
Unknown
SPR_52920 182 AHF81552 Streptomyces netropsis DSM
40093
65 Nucleoside 2-deoxyribosyltransferas
e
Unknown
SPR_52930 319 AHF81551 Streptomyces netropsis DSM
40093
73 Dihydroorotate dehydrogenase
Unknown
SPR_52940 266 AHF81550 Streptomyces netropsis DSM
40093
79 Creatinine amidohydrolase
Unknown
SPR_52950 377 AHF81549 Streptomyces netropsis DSM
40093
79 Hypothetical protein -hydrolase
Unknown
SPR_52960 437 AHF81548 Streptomyces netropsis DSM
40093
75 Nucleotide sugar dehydrogenase
Unknown
SPR_52970 330 AHF81547 Streptomyces netropsis DSM
40093
78 Nucleoside-diphosphate sugar
epimerase
Unknown
SPR_52980 354 AHF81546 Streptomyces netropsis DSM
40093
72 Glycosyltransferase Unknown
SPR_52990 254 CAJ88617 Streptomyces ambofaciens ATCC
23877
73 Sugar nucleotidyl transferase
Unknown
SPR_53000 383 AHF81544 Streptomyces netropsis DSM
40093
77 Nucleotide sugar aminotransferase
Unknown
SPR_53010 641 AHF81543 Streptomyces netropsis DSM
40093
71 Mannanase Unknown
Chapter 3 – Streptomyces polyantibioticus SPRT genome exploration
123
SPR_53020 287 AHF81561 Streptomyces netropsis DSM
40093
76 Amidohydrolase Unknown
SPR_53030 271 AHF81542 Streptomyces netropsis DSM
40093
70 Methyltransferase Unknown
SPR_53040 450 AIS24860 Streptomyces netropsis DSM
40093
60 NRPS, C domain DPO assembly
SPR_53050 348 AHF81540 Streptomyces netropsis DSM
40093
78 Alcohol dehydrogenase
Unknown
SPR_53060 1084
AHF81539 Streptomyces netropsis
DSM40093
60 NRPS, A-PCP-C DPO assembly
SPR_53070 124 ASI24864 Streptomyces netropsis
DSM40093
66 NRPS, PCP domain Not involved
SPR_53080 221 WP_037790144 Streptomyces sp.
Mg1
72 Hypothetical protein Not involved
SPR_53090 259 WP_030674398
Streptomyces sp. NRRL B-1347
79 TE domain DPO assembly
NRPS, nonribosomal peptide synthetase; C, condensation; A, adenylation; PCP, peptidyl carrier protein; TE,
thioesterase. Percentages of identity refer to deduced amino acid sequence comparisons.
Chapter 3 – Streptomyces polyantibioticus SPRT genome exploration
124
Figure 3.3 Genetic organization of the putative DPO biosynthetic gene cluster.
Furthermore, in order to further characterize the individual NRPS domains contained within
the putative DPO biosynthetic cluster, known signature motifs and catalytic residues were
identified and assessed for integrity against a selection of reference proteins derived from the
CDD (Marchler-Bauer et al., 2011). These analyses are described in the following sections.
3.4.4.1 A DOMAIN SPECIFICITY
Prediction of the substrate specificity of the A domains encoded by SPR_52860 and
SPR_53060 was carried out using the NRPSpredictor2 (Röttig et al., 2011), the LSI algorithm
(Baranasic et al., 2013), NRPS/PKS predictor (Bachmann & Ravel, 2009), the Non-Ribosomal
Peptide Synthase Substrate Predictor (NRPSsp) (Prieto et al., 2012) and the Stachelhaus
specificity-conferring code (Stachelhaus et al., 1999) (Table 3.3). The A domain specificity
predictions were not conclusive as the web-based NRPS prediction services produced different
Chapter 3 – Streptomyces polyantibioticus SPRT genome exploration
125
results for the A domains encoded by SPR_52860 and SPR_53060. However, the A domain
specificity results for the domains encoded by SPR_52860 and SPR_53060 were in agreement
with the prediction that the substrate would be aromatic i.e. tryptophan, phenylalanine and/or
2,3-dihydroxybenzoic acid.
The NRPSpredictor2 result for the query domain encoded by SPR_52860, based on the gross
physico-chemical properties, was that the substrate is hydrophobic and aliphatic, but with a
very low score of 0.190785. The result based on the gross physico-chemical properties for the
query domain encoded by SPR_53060, predicts that the substrate is hydrophilic with a slightly
higher, but still inconclusive score of 0.375341. On the basis of these highly questionable
predictions, it was not possible to infer the A domain specificity of the query domains encoded
by SPR_52860 and SPR_53060 using the NRPSpredictor2 output. Furthermore, Rausch et al.
(2005) commented that the NRPSpredictor2 server predicts aromatic substrates less reliably
due to the observed promiscuity of the A domains utilizing these substrates (Rausch et al.,
2005). Indeed, substrate promiscuity has been observed in Xenorhabdus nematophila where
two A domains that exhibit specificity for tryptophan in xenematide A biosynthesis can accept
either tryptophan or phenylalanine as substrates to produce a diverse family of products.
Markedly, this tryptophan/phenylalanine promiscuity is accommodated in the downstream
domains and results in a small family of cyclic depsipeptides (Crawford et al. 2011).
Instances of relaxed substrate specificity have also been observed in cyanobacterial NRPS A
domains, where structurally related amino acids such as valine, isoleucine and leucine are
located in equivalent positions of the final peptide products. These NRPS pathways produce
entire families of structurally related compounds all co-occurring in one specific isolate
(Christiansen et al., 2011).
Both of the A-domain specificity prediction results obtained from the LSI algorithm were based
on a LSI score of below 0.66, which is classified as an unreliable prediction (Baranasic et al.,
2013).
The A domain specificity prediction based on the profile Hidden Markov Model (pHMM)
approach developed by Prieto et al. (2012) and known as NRPSsp, resulted in both query
domains predicted to be specific for the activation of phenylalanine. However, the authenticity
of this prediction server was questioned by Khayatt et al. (2013) who claimed that the dataset
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126
that the NRPSsp server is based on contains erroneous annotations and several sequences not
related to NRPSs.
The NRPS/PKS prediction server developed by Bachmann & Ravel (2009) could not identify
the A domain specificity for either query domain. The Stachelhaus specificity-conferring code
predicted the substrates 2,3-dihydroxybenzoic acid and tryptophan for the query domains
encoded by SPR_52860 and SPR_53060, respectively, with a score of 60 % in each case. In
comparison to the scores achieved by the other prediction servers/algorithms, these are the most
reliable. However, Rausch et al. (2005) noted that Stachelhaus predictions at less than a 70%
threshold are less reliable and lead to inconsistent predictions. In light of this statement and
the fact that tryptophan-and phenylalanine-specific domains are known to be interchangeable,
it is possible that the query domain, SPR_53060, displays a relaxed specificity and activates
phenylalanine (or 3-hydroxyphenylalanine) instead of tryptophan. Furthermore, it is possible
that the query domain, SPR_52860, also displays relaxed substrate specificity, allowing it to
activate benzoic acid rather than 2,3-dihydroxybenzoic acid.
Table 3.3 A domain specificity predictions
NRPSpredictor 2
(Röttig et al.,
2011)
LSI
(Baranasic et
al., 2013)
NRPS/PKS
(Bachmann &
Ravel, 2009)
NRPSsp
(Prieto et al.,
2012)
Stachelhaus
code
(Stachelhaus
et al., 1999)
SPR_52860 Hydrophobic-aliphatic
tryptophan NO HIT phenylalanine 2,3-dihydroxy-benzoic acid
SPR_53060 Hydrophilic tryptophan NO HIT phenylalanine tryptophan
3.4.4.2 CY DOMAINS
The NaPDos online tool did not identify any obvious Cy domains within the S. polyantibioticus
SPRT genome sequence. In light of this, a phylogenetic analysis was performed in order to
determine the evolutionary relatedness of all the annotated C domains within the S.
polyantibioticus SPRT genome in comparison to reference Cy domain sequences from the
Streptomyces virginiae NRRL B-1446T VirH sequence (GenBank accession number:
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AB283030), S. coelicolor A3(2) putative non-ribosomal peptide synthetase sequence
(CAC17500), Streptomyces verticillatus strain ATCC 15003 BlmIV sequence (AAG02364),
Pseudomonas aeurginosa PA14 PchE sequence (AAD55800), Bacillus licheniformis ATCC
10716 BacA sequence (O68006), Sorangium cellulosum So ce90 EposP sequence (AAF26925)
and the Streptomyces flavoviridis ATCC 21892 Zbm sequence (EU670723).
The phylogenetic analysis, using the neighbour joining (NJ) (Figure 3.4) and maximum
likelihood (ML) (Figure 3.5) algorithms, resolved the putative C domains from
S. polyantibioticus SPRT and the reference Cy domain sequences into two distinct groups. The
first group consisted of 19 of the putative C domains found within the S. polyantibioticus SPRT
genome sequence, while the second group consisted of all of the reference Cy domains plus
the S. polyantibioticus SPRT C domains encoded by SPR_52900, SPR_53040, SPR_53060,
SPR_6230, SPR_6240 and SPR_6360. From the neighbour-joining phylogenetic analysis, it
can be inferred that the S. polyantibioticus SPRT genes in the second group are further sub-
divided into three clusters, wherein SPR_6230 was more closely related to the reference Cy
domains than SPR_6360, SPR_6240, SPR_53040, SPR_53060, and SPR_52900. Moreover,
SPR_6240 was more closely related to SPR_53040 and SPR_53060 than was SPR_6230, with
SPR_52900 being the least related to the reference Cy domains. However, the bootstrap support
for the association of SPR_6230, SPR_6240, SPR_6360, SPR_53040 and SPR_53060 with
known Cy domain sequences was very weak (<50%).
In contrast, the phylogenetic analysis using the ML algorithm resolved the S. polyantibioticus
SPRT genes in the second group into five clusters, wherein SPR_6240 was the most closely
related to the reference Cy domains, followed by SPR_53040, SPR_53060, SPR_6360,
SPR_52900 and SPR_6230. Similarly to the NJ tree, the bootstrap support for the association
of SPR_6230, SPR_6240, SPR_6360, SPR_52040 and SPR_53060 with known Cy domain
sequences was very weak (<50%).
Similarly, the phylogenetic analysis using the maximum parsimony (MP) (Figure 3.6)
algorithm, resolved the C domains from S. polyantibioticus SPRT and the reference Cy domain
sequences into two distinct groups. However, while the first, most distantly-related group
consisted of only S. polyantibioticus SPRT encoded C domains, the second group consisted of
the Cy reference domains, in addition to the S. polyantibioticus SPRT genes encoded by
SPR_6230, SPR_6240, SPR_6360, SPR_53060 and SPR_53040. This algorithm established
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that SPR_6360 was most closely related to the reference Cy domains, followed by SPR_6240,
SPR_53060, SPR_53040 and SPR_6230. In contrast to the NJ and ML algorithms, SPR_52900
was not associated with the Cy domain reference sequences in this instance.
It is clear from the phylogenetic analyses that the S. polyantibioticus SPRT amino acid
sequences from SPR_6230, SPR_6240, SPR_6360, SPR_53060 and SPR_53040 were
consistently associated with the reference Cy domains, suggesting that SPR_6230, SPR_6240,
SPR_6360, SPR_53060 and SPR_53040 encode Cy domains, despite being annotated as C
domains. It has been reported that despite secondary structure predictions indicating a similar
overall fold for both Cy and C domains, there is weak primary sequence homology between
the two (Kelly et al., 2005; Keating et al., 2002). This has contributed to the lack of knowledge
regarding the catalytic mechanism of the Cy domains and only a detailed structural and
mutational analysis of the catalytic residues of the domains will help to elucidate the
mechanisms involved (Hur et al., 2012). Despite primary sequence differences, Figures 3.3-
3.5 show that C and Cy domains can be distinguished by phylogenetic analyses.
The phylogenetic analysis of the putative C and Cy domains prompted a comprehensive
investigation of the signature motifs and catalytic residues contained within the domains
encoded by SPR_53060, SPR_52900, SPR_53040, SPR_6230, SPR_6240 and SPR_6360, as
these domains shared the highest degree of evolutionary relatedness to the reference Cy
domains (Figure 3.7).
Heterocyclization domains replace C domains in NRPS modules that produce thiazoline or
oxazoline rings. In addition, the catalytic core motif, H-H-x-x-x-D-G, of regular C domains is
replaced by a D-x-x-x-x-D-x-x-S motif found in Cy domains; the two aspartate residues play
an essential part in both condensation and heterocyclization reactions (Keating et al., 2002).
An additional seven Cy domain signature motifs were described by Schwarzer et al. (2003), all
of which were analysed using a multiple sequence alignment with reference Cy domain
sequences from the S. virginiae NRRL B-1446T VirH sequence (GenBank accession number:
AB283030), S. coelicolor A3(2) putative non-ribosomal peptide synthetase sequence
(CAC17500), S. verticillatus strain ATCC 15003 BlmIV sequence (AAG02364),
Pseudomonas aeurginosa PA14 PchE sequence (AAD55800), Bacillus licheniformis ATCC
10716 BacA sequence (O68006), Sorangium cellulosum So ce90 EposP sequence (AAF26925)
and the Streptomyces flavoviridis ATCC21892 Zbm sequence (EU670723) (Figure 3.7).
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The putative Cy domains encoded by SPR_53040, SPR_53060 and SPR_52900 shared a
35 %, 41 % and 24 % homology, respectively, to the eight NRPS Cy domain signature motifs.
By comparison, the two Cy domains encoded by the angN gene found in the NRPS module of
Vibrio anguillarum 775, revealed only 23 % and 19 % homology with 7 other characterised
Cy domains in a multiple sequence alignment (Di Lorenzo et al., 2008). Within the core motif
(D-x-x-x-x-D-x-x-S), SPR_52900 displayed neither of the essential catalytic aspartate residues
and was therefore concluded to be inactive. In contrast, SPR_6230 displayed both of the
critical catalytic aspartate residues, while SPR_6240 and SPR_6360 each displayed only the
second catalytic aspartate residue. Similarly, SPR_53040 and SPR_53060 each displayed only
one of the catalytic aspartate residues. However, SPR_53040 displayed the first catalytic
aspartate residue, while SPR_53060 displayed the second aspartate residue, suggesting the
possible complementation of the catalytic activity required in the formation of DPO.
Indeed, it has been demonstrated that the two different Cy domains found in the VibF NRPS
of the vibriobactin biosynthetic pathway perform either the role of the condensation reaction
or the role of heterocyclization. A mutation of the catalytic aspartate residue in one domain
resulted in lower heterocyclic product formation, whilst maintaining condensation activity. A
mutation in the second domain caused the opposite effect (Keating et al., 2002). However,
from the multiple sequence alignment alone, it cannot be determined whether the two domains,
SPR_53040 and SPR_53060, together provide the catalytic activity required for the formation
of DPO by S. polyantibioticus SPRT.
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Figure 3.4 Unrooted phylogenetic tree obtained by the neighbour-joining method (Saitou & Nei, 1987) from an alignment of the amino acid sequences of S. polyantibioticus SPRT C domains and heterocyclization domain sequences from the S. virginiae NRRL B-1446T Vir sequence (S.virginiae cyc), S. coelicolor A3(2) putative non-ribosomal peptide synthetase sequence (S. coelicolor cyc), S. verticillatus strain ATCC 15003 BlmIV sequence (S.verticillus cyc), Pseudomonas aeurginosa PA14 PchE sequence (P.aeurginosa cyc), Bacillus licheniformis ATCC 10716 BacA sequence (B. licheniformis cyc), Sorangium cellulosum So ce90 EposP sequence (S. cellulosum cyc) and the Streptomyces flavoviridis ATCC21892 Zbm sequence (S.flavoridis cyc). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches and the GenBank accession numbers are displayed in parenthesis. The analysis involved 32 amino acid sequences and all positions containing gaps and missing data were eliminated. Scale bar, 20 amino acid substitutions per 100 amino acids.
Chapter 3 – Streptomyces polyantibioticus SPRT genome exploration
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Figure 3.5 Unrooted phylogenetic tree obtained by the Maximum Likelihood method (Jones et al., 1992) based on the JTT matrix-based model for the amino acid sequences of S. polyantibioticus SPRT C domains and heterocyclization domain sequences from the S. virginiae NRRL B-1446T VirH sequence (S.virginiae cyc), S. coelicolor A3(2) putative non-ribosomal peptide synthetase sequence (S. coelicolor cyc), S. verticillatus strain ATCC 15003 BlmIV sequence (S.verticillus cyc), Pseudomonas aeurginosa PA14 PchE sequence (P.aeurginosa cyc), Bacillus licheniformis ATCC 10716 BacA sequence (B. licheniformis cyc), Sorangium cellulosum So ce90 EposP sequence (S. cellulosum cyc) and the Streptomyces flavoviridis ATCC21892 Zbm sequence (S.flavoridis cyc). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches and the GenBank accession numbers are displayed in parenthesis. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value (Tamura et al., 2013). The analysis involved 32 amino acid sequences. All positions containing gaps and missing data were eliminated. Scale bar, 50 amino acid substitutions per 100 amino acids.
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Figure 3.6 Unrooted phylogenetic tree obtained by the Maximum Parsimony (MP) method (Felsenstein, 1985) for the amino acid sequences of S. polyantibioticus SPRT C domains and heterocyclization domain sequences from the S. virginiae NRRL B-1446T VirH sequence (S.virginiae cyc), S. coelicolor A3(2) putative non-ribosomal peptide synthetase sequence (S. coelicolor cyc), S. verticillatus strain ATCC 15003 BlmIV sequence (S.verticillus cyc), Pseudomonas aeurginosa PA14 PchE sequence (P.aeurginosa cyc), Bacillus licheniformis ATCC 10716 BacA sequence (B. licheniformis cyc), Sorangium cellulosum So ce90 EposP sequence (S. cellulosum cyc) and the Streptomyces flavoviridis ATCC21892 Zbm sequence (S.flavoridis cyc). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches and the GenBank accession numbers are displayed in parenthesis. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates) (Nei & Kumar, 2000). The analysis involved 32 amino acid sequences. All positions containing gaps and missing data were eliminated.
Chapter 3 – Streptomyces polyantibioticus SPRT genome exploration
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Cyc motif 1 F P L T/S 2x Q x A Y 2x G R VirH-cyc L P L T (2x) Q S A Y (2x) G R NRPS-cyc F P L T (2x) Q A A Y (2x) G R blmIV-cyc F P L T (2x) Q R A Y (2x) G R BacA-cyc F P L T (2x) Q L A Y (2x) G R EposP-cyc F P L T (2x) Q E S Y (2x) G R PchE-cyc F E L S (2x) Q Q A Y (2x) G R Zbm-cyc F P L T (2x) Q Q A Y (2x) G R SPR_6230 A P P S (2x) Q E E H (2x) Q A SPR_6240 A P L S (2x) Q E E M (2x) N I SPR_6360 G P L S (2x) Q R R F (2x) A E SPR_53040 G P L S (2x) Q L G M (2x) L E SPR_53060 - P L T (2x) Q W R M (2x) H H SPR_52900 A S A T (2x) Q R Q M (2x) L I
Cyc motif 2 R H P/A/L G x Q Cy motif 3 D 4x D 2x S VirH-cyc H H R V H Q VirH-cyc D (4x) D (2x) S NRPS-cyc R H R I H Q NRPS-cyc D (4x) D (2x) S blmIV-cyc R H R V L P blmIV-cyc D (4x) D (2x) S BacA-cyc R H R V F E BacA-cyc D (4x) D (2x) S EposP-cyc R H R T L P EposP-cyc D (4x) D (2x) S PchE-cyc R H R F F D PchE-cyc D (4x) D (2x) S Zbm-cyc R H R I D A Zbm-cyc D (4x) D (2x) S SPR_6230 R H R F R T SPR_6230 D (4x) D (2x) S SPR_6240 R H R I A Y SPR_6240 H (4x) D (2x) S SPR_6360 R H R Y P W SPR_6360 H (4x) D (2x) S SPR_53040 R H R F T V SPR_53040 D (4x) W (2x) V SPR_53060 R H R L A T SPR_53060 H (4x) D (2x) S SPR_52900 R H E S L R SPR_52900 S (4x) A (2x) S
Cy motif 4 L P 2x P x L P L 3x P VirH-cyc L P (2x) P P L P D (3x) Q NRPS-cyc L P (2x) P E L P L (3x) A blmIV-cyc L P (2x) P G L P L (3x) P BacA-cyc F P (2x) P E L P L (3x) P EposP-cyc L P (2x) P T L P M (3x) P PchE-cyc L P (2x) P A L P L (3x) P Zbm-cyc L P (2x) P E L P F (3x) P SPR_6230 L S (2x) L L P Q A (3x) P SPR_6240 L A (2x) T G E V A (3x) P SPR_6360 A A (2x) P V L P T (3x) N SPR_53040 V A (2x) P E L T L (3x) T SPR_53060 A P (2x) T R L P T (3x) P SPR_52900 A P (2x) M F P G R (3x) G
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Cy motif 5 T/S P/A 3x L/A/F 6x I/V/T L 2x W VirH-cyc P A 3x L 6x E L 2x T NRPS-cyc T P 3x L 6x T V 2x W blmIV-cyc T P 3x I 6x V L 2x W BacA-cyc T P 3x L 6x I L 2x W EposP-cyc T P 3x I 6x V I 2x W PchE-cyc T L 3x F 6x V L 2x W Zbm-cyc T P 3x L 6x I V 2x W SPR_6230 T L 3x L 6x A S 2x I SPR_6240 S P 3x L 6x A I 2x V SPR_6360 A T 3x C 6x G R 2x P SPR_53040 T L 3x L 6x V L 2x H SPR_53060 T P 3x I 6x V A 2x S SPR_52900 S T 3x L 6x L L 2x R
Cy motif 6 G/A D F T x L x L L VirH-cyc A D F T Q L A W V NRPS-cyc G D F T S L E L L blmIV-cyc G D F T T T T L L BacA-cyc G D F T S L M L L EposP-cyc G D F T S M V L L PchE-cyc A D F T T L L L L Zbm-cyc G D F T S L I P L SPR_6230 G W Y I N A A P I SPR_6240 G H L L N T R L T SPR_6360 V V D T A A D Q V SPR_53040 G L F V N M L P I SPR_53060 G L F T H T V P L SPR_52900 A N L H Q E V L T
Cy motif 7 P V V F T S x L VirH-cyc P V V F T R V P NRPS-cyc P V V F T S A L blmIV-cyc P V V F T S T L BacA-cyc P I V F T S V L EposP-cyc P V V L T S A L PchE-cyc P V V F A S N L Zbm-cyc P V V F T S N L SPR_6230 S Y L D F R P L SPR_6240 L S P E W E A Q SPR_6360 S S V R V R P P SPR_53040 I N V C V S F Q SPR_53060 Q M V F C H G A SPR_52900 T V V A S S D F
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Cy motif 8 S/T Q/R T P Q V x L/I D 13x W D VirH-cyc S Q T A Q V A L D 13x W D NRPS-cyc T R T P Q V W L D 13x W D blmIV-cyc S Q T P Q V L L D 13x W D BacA-cyc T R T S Q V Y I D 13x W D EposP-cyc T Q T P Q L L L D 13x W D PchE-cyc S Q T P Q V W L D 13x W D Zbm-cyc - - - - - - - - - - - - SPR_6230 S G T G I D L F L 13x P D SPR_6240 S W R S F A V L W 13x R P SPR_6360 Q W R E D V M D R 13x A E SPR_53040 S P F D L D L G F 13x N P SPR_53060 A K F D V T V M V 13x Y D SPR_52900 C A E T A A L S P 13x L V
Figure 3.7 A multiple sequence alignment of the conserved signature motifs of the putative S. polyantibioticus SPRT Cy domains, encoded by SPR_6230, SPR_6240, SPR_6360, SPR_53040, SPR_53060 and SPR_52900, and reference Cy domain amino acid sequences taken from the S. virginiae NRRL B-1446T VirH sequence (VirH-cyc) (AB283030), S. coelicolor A3(2) putative non-ribosomal peptide synthetase sequence (NRPS-cyc) (CAC17500), S. verticillatus strain ATCC 15003 BlmIV sequence (blmIV-cyc) (AAG02364), Pseudomonas aeurginosa PA14 PchE sequence (PchE-cyc) (AAD55800), Bacillus licheniformis ATCC 10716 BacA sequence (BacA-cyc) (O68006), Sorangium cellulosum So ce90 EposP sequence (EposP-cyc) (AAF26925) and the Streptomyces flavoviridis ATCC21892 Zbm sequence (Zbm-cyc) (EU670723). Residues critical for the domain function are coloured green while other residues corresponding to the consensus motif are coloured yellow.
3.4.4.3 PCP DOMAINS
The peptidyl carrier protein enables the transfer of activated amino acids and elongation
intermediates between catalytic centres. The PCP domain is characterised by a conserved
serine residue, which accepts a phosphopantetheine moiety as a prosthetic group, thereby
converting the inactive apo-PCP to its active holo-form. A variant of the PCP has been
identified in the synthetases of siderophores and other aryl-capped non-ribosomal peptides for
the incorporation of aromatic carboxy acids, where aryl acids such as salicylate and 2,3-
dihydroxybenzoic acid are tethered on ArCPs (Crosa & Walsh, 2002). In particular,
freestanding A domains are known to activate aromatic carboxylic acids and transfer them to
aryl-carrier proteins (ArCP), which are used as starter substrates in, for example, quinoxaline
antibiotics (Crawford et al., 2011).
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Marahiel et al. (1997) identified the signature motif, L-G-G-(D/H)-S-L, in all PCP and ArCP
domains. The antiSMASH analysis of the draft S. polyantibioticus SPRT genome identified
two PCP domains in the putative DPO gene cluster (Table 3.2), which were examined in a
multiple sequence alignment with reference ArCP domains from Yersinia enterocolitica subsp.
enterocolitica 8081 (GenBank accession number: YE2617), Brucella melitensis bv. 1 str. 16M
(BMEII0079), Bacillus subtilis (BSU31970), Escherichia coli (P0ADI4), Pseudomonas
aeruginosa PA14 (AAD55800), Salmonella enterica subsp. enterica (STM0597) and
Streptomyces pyridomyceticus (AEF33099). The alignment revealed the presence of the
critical catalytic serine residue in the putative ArCP domain encoded by SPR_53060, and the
absence of this serine residue in the putative PCP domain encoded by SPR_53070 (Figure 3.8).
The signature motif is well conserved in the SPR_53060 domain and very poorly conserved in
the SPR_53070 domain, with the exception of the final leucine residue, suggesting the possible
inactivity/redundancy of the domain encoded by SPR_53070. Indeed, the essential serine
residue is replaced by an arginine residue, which would prevent the attachment of the
phosphopantetheine prosthetic group.
PCP motif L G G D/H S L Y.enterocolitica-ArCP A G L D S I B.melitensis-ArCP Y G L D S L B.subtilis-ArCp R G L D S V E.coli-ArCp Y G L D S V P.aeruginosa-ArCP C G L D S I S.enteric-ArCP Y G L D S V S.pyridomyceticus-ArCP L G L D S I SPR_53060 R G G D S L SPR_53070 Q A I G R L
Figure 3.8 A multiple sequence alignment of the conserved signature motifs of the putative S. polyantibioticus SPRT ArCP domain encoded by SPR_53060 and the PCP domain encoded by SPR_53070 with reference ArCP domains from Yersinia enterocolitica subsp. enterocolitica 8081 (Y.enterocolitica-ArCP), Brucella melitensis bv. 1 str. 16M (B.melitensis-ArCP), Bacillus subtilis (B.subtilis-ArCP), Escherichia Coli (E.coli-ArCP), Pseudomonas aeruginosa PA14 (P.aeruginosa-ArCP), Salmonella enterica subsp. enterica (S.enteric-ArCP)) and Streptomyces pyridomyceticus (S.pyridomyceticus-ArCP). Residues critical for the domain function are coloured green while other residues corresponding to the consensus motif are coloured yellow.
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Due to the fact that both PCP and ArCP domains share the same conserved signature motif, a
phylogenetic analysis was performed (Figure 3.9) in order to determine the evolutionary
relatedness of the putative ArCP domain encoded by SPR_53060 and the PCP domain encoded
by SPR_53070 to the reference ArCP domains used in the analysis of the conserved signature
motifs (Figure 3.8). The PCP domains from Streptomyces netropsis DSM 40846 (GenBank
accession number: AHF81539), Streptomyces sp. CNS615 (WP_037734274), Streptomyces
himastatinicus ATCC 53653 (WP_009720528), S.coelicolor A3(2) (NP_631722),
Streptomyces lividans 1326 (EOY52612) were added to the phylogenetic analysis.
The phylogenetic analysis, using the NJ, ML and MP algorithms, resolved the putative ArCP
and PCP domains from S. polyantibioticus SPRT and the reference sequences into two distinct
groups. The reference ArCP domains clustered together in all three trees (with very high
bootstrap support (98 %) in the NJ tree) and the reference PCP domains clustered together with
the amino acid sequences of SPR_53060 and SPR_53070. There was strong bootstrap support,
93 % and 67 %, respectively, for the association of SPR_53060 and SPR_53070 with known
PCP domain sequences.
This analysis indicated that the domain encoded by SPR_53060 is in fact a PCP domain and
not an ArCP, as originally suggested by the conserved signature motif analysis.
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Figure 3.9 Unrooted, neighbour joining tree (Saitou & Nei, 1987) obtained from an alignment of the amino
acid sequences of the putative S. polyantibioticus SPRT ArCP domain encoded by SPR_53060 and the PCP domain encoded by SPR_53070 with reference ArCP domains from Yersinia enterocolitica subsp. enterocolitica 8081 (Y.enterocolitica-ArCP), Brucella melitensis bv. 1 str. 16M (B.melitensis-ArCP), Bacillus subtilis (B.subtilis-ArCP), Escherichia Coli (E.coli-ArCP), Pseudomonas aeruginosa PA14 (P.aeruginosa-ArCP), Salmonella enterica subsp. enterica (S.enterica-ArCP)) and Streptomyces pyridomyceticus (S.pyridomyceticus-ArCP), in addition to PCP domains from Streptomyces netropsis DSM 40846 (S.netropsis PCP), Streptomyces sp. CNS615 (Streptomyces sp. CNS615 PCP), Streptomyces himastatinicus ATCC 53653 (S.himastatinicus PCP), S.coelicolor A3(2) (S.coelicolor PCP) and Streptomyces lividans 1326 (S.lividans PCP). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches and the GenBank accession numbers are displayed in parenthesis. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site (Zuckerkandl & Pauling, 1965). The analysis involved 14 amino acid sequences and all positions containing gaps and missing data were eliminated. Asterisks indicate clades that were conserved in neighbour-joining, maximum-likelihood and maximum-parsimony trees. Scale bar, 20 amino acid substitutions per 100 amino acids.
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3.4.4.4 OX DOMAINS
An Ox domain is required during DPO biosynthesis for the oxidation of the initial unstable
oxazoline ring to an oxazole ring, as postulated in Chapter 2 (section 2.2.1). Ox domains have
been identified as members of the McbC-like oxidoreductase superfamily and are normally
associated with heterocyclization modules (Marchler-Bauer et al., 2011).
A BLASTP search against the S. polyantibioticus SPRT draft genome, using well-characterised
Ox domain amino acid sequences from: Streptomyces hygroscopicus McbC-like
oxidoreductase (GenBank accession number: ACS50132), Streptomyces sp. NRRL WC-3773
NADH oxidase (WP_031005407), Streptomyces verticillus ATCC15003 peptide synthetase
(AAG02365), S. cellulosum epothilone biosynthetic cluster (AAF62881) and Angiococcus
disciformis tubulysin biosynthetic cluster (CAF05649), revealed the existence of putative Ox
domains within the genome encoded by the genes SPR_01300, SPR_37410, SPR_39290,
SPR_39400, SPR_44390, SPR_49120 and SPR_53180 amongst others. However, no Ox
domains were found within the putative DPO biosynthetic cluster (section 3.4.4). This suggests
that the oxidation activity required in DPO biosynthesis may be provided by an external
tailoring enzyme. Indeed, the in trans thiazoline to thiazole oxidase activity of the S.
cellulosum EpoB Ox domain was demonstrated by Schneider et al. (2003) who heterologously
expressed this domain in E. coli. Additionally, the mechanism of the oxidation process remains
unclear, but it does share a clear analogy to the oxidation of dihydroorotate to orotate observed
in pyrimidine biosynthesis, in which an FMN co-factor-mediated redox step is involved
(Schenider et al., 2003). In light of this, it is interesting to note that a homologue of the key
enzyme involved in this reaction, dihydroorotate dehydrogenase, is encoded by the gene
SPR_52930, identified as a member of the putative DPO biosynthetic pathway. Although DPO
is not a pyrimidine, both compounds are aromatic heterocycles and therefore the presence of a
homologue of dihydroorotate dehydrogenase within the putative DPO biosynthetic cluster may
indicate its involvement in the oxidation process required for DPO biosynthesis.
Lastly, in contrast to all other NRPS domains, whose relative position within a given module
is generally highly conserved, the location of the EpoB and BlmIII Ox domains from
S. cellulosum and Streptomyces verticillatus ATCC 15003, respectively, suggest that Ox
domains can be located either upstream or downstream of the PCP domains presenting
thiazolinyl-S-pantotheinyl chains for oxidation. This may be indicative of the fact that Ox-PCP
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and PCP-Ox pairs may be transferable to other Cy-containing modules in NRPS assembly
lines, thereby preserving the ability to direct the catalysis of thiazolinyl and oxazolinyl-S-
enzyme intermediates to heterocycle oxidation states (Schneider et al., 2003). A similar
atypical positioning of the Ox domain may also occur in the genes for DPO biosynthesis in
S. polyantibioticus SPRT.
3.4.4.5 TE DOMAIN
The gene SPR_53090 was identified by antiSMASH to encode a TE domain. These domains
share the same G-x-S-x-G motif, described by Schwarzer et al. (2003), as the acyltransferase
enzymes found in PKS systems that catalyse the transfer of acyl moieties between CoA and
ACP using a serine-histidine catalytic diad (Yadav et al., 2003).
A multiple sequence alignment of SPR_53090 with known TE domains from Bacillus and
Streptomyces species revealed the highly conserved G-x-S-x-G signature motif in SPR_53090
(Figure 3.10), thereby indicating that it is likely responsible for the thioesterase activity in the
proposed DPO biosynthetic gene cluster.
Te motif G x S x G B.cereus-Te G H S M G B.licheniformis-Te G H S M G S.coelicolor-Te G H S M G S.aureofaciens-Te G H S M G SPR_53090 G H S L G
Figure 3.10 A multiple sequence alignment of the conserved signature motif of the putative DPO TE domain from S. polyantibioticus SPRT and various TE reference sequences taken from S. coelicolor A3(2) (S.coelicolor-Te) (GenBank ccession number: NP_631726), S. aureofaciens (S.aureofaciens-Te) (YP_009060793), Bacillus cereus AH1134 (B.cereus-Te) (EDZ49042) and B. licheniformis IBL200 (B.licheniformis-Te) (AAU22005). Residues critical for the domain function are coloured green while other residues corresponding to the consensus motif are coloured yellow.
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3.4.4.6 GENES INVOLVED IN BENZOIC ACID BIOSYNTHESIS
In addition to identifying the NRPS-associated genes involved in DPO biosynthesis, the genes
for benzoic acid biosynthesis also need to be identified. Despite its common occurrence in
plant metabolites, benzoic acid biosynthesis is not common in bacteria (Moore et al., 2002).
However, the biosynthesis of benzoate as a starter unit in the biosynthesis of enterocin in
‘Streptomyces maritimus‘ strain DSM 41777T prompted a search of the draft S. polyantibioticus
SPRT genome for an orthologue of the encP gene, encoding the key enzyme, phenylalanine
ammonia-lyase. An encP homologue is not present in the putative DPO biosynthetic cluster,
or anywhere else in the S. polyantibioticus SPRT genome.
The annotation of the draft genome of S. polyantibioticus SPRT revealed the presence of an
enoyl-CoA hydratase, cinnamate-CoA ligase, and an acyl-CoA dehydrogenase encoded by
genes SPR_60140, SPR_60150 and SPR_60160, respectively. The enoyl-CoA hydratase and
cinnamate CoA ligase shared 25 % and 27 % homology, respectively, to the homologous
proteins found in the benzoate biosynthetic pathway identified in ‘S. maritimus‘ strain DSM
41777T.
Despite the fact that a PAL homologue was not identified in the S. polyantibioticus SPRT draft
genome, an alternative route to cinnamic acid was proposed whereby phenylalanine would be
catabolized to cinnamic acid via phenylpyruvate and phenyllactate (Figure 3.9). Indeed,
several catabolic pathways for L-phenylalanine have been reported in various organisms,
including Streptomyces setonii 75Vi2, which metabolises L-phenylalanine via phenylpyruvate
and phenyllactate (Pometto III & Crawford, 1985). Moreover, it has been reported that lactic
acid bacteria catabolize phenylalanine to phenylpyruvate via an aminotransferase, after which
phenylpyruvate is catabolized to either D- or L-phenyllactate via dehydrogenase activity (Mu
et al., 2012). It has also been demonstrated that a D-lactate dehydrogenase from Leuconostoc
mesenteroides can reduce phenylpyruvate to phenyllactate (Simon et al., 1989).
In light of these reports and the fact that a putative aminotransferase encoded by SPR_60250
and a putative D-lactate dehydrogenase encoded by SPR_60260 were clustered together with
the genes encoding the enoyl-CoA hydratase, cinnamate-CoA ligase, and an acyl-CoA
dehydrogenase mentioned above, it was decided to explore the possibility of these genes being
involved in the biosynthesis of benzoate and therefore DPO production, as functionally related
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genes are usually clustered in bacterial genomes (Kaneko et al., 2003). Therefore, the genes
encoding the cinnamate-CoA ligase and D-lactate dehydrogenase were identified as putative
external constituents of the DPO biosynthetic pathway. The investigation into their
involvement in the DPO biosynthetic pathway is explored in Chapter 4.
Figure 3.9 Proposed biosynthetic pathway for benzoyl-CoA production in S. polyantibioticus SPRT.
Lastly, the gene coding for the phenylacetate-coenzyme A ligase, paaK, which was identified
in the S. polyantibioticus SPRT genome via PCR amplification and sequencing (Chapter 2),
was identical to the sequence of the gene SPR_46390 obtained from the annotation of the draft
S. polyantibioticus SPRT genome. The antiSMASH 3.0 (Weber et al., 2015) analysis identified
the cluster that the paaK gene was located in as a PKS/NRPS hybrid encoding an antimycin-
type antibiotic. The composition of the gene cluster was most similar in gene content and gene
order to the antimycin biosynthetic gene cluster from Streptomyces blastmyceticus, as 100 %
of the S. polyantibioticus SPRT genes shared homology to genes within this cluster but, due to
the complicated structure of antimycin (PubChem ID: 14957) (Figure 3.10), it was deemed
unlikely that this cluster would be involved in DPO biosynthesis. However, further efforts to
characterize the involvement of paaK in the synthesis of DPO are described in Chapter 4.
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Figure 3.10 Outline of the S. polyantibioticus SPRT antimycin structure predicted by antiSMASH 3.0.
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3.5 CONCLUSION
The draft S. polyantibioticus SPRT genome sequence was obtained using a hybrid sequencing
approach, after which the sequence was annotated and secondary metabolite gene clusters were
identified using antiSMASH 3.0 analysis (Weber et al., 2015). The putative DPO biosynthetic
gene cluster was identified using a genome mining approach in which each of the six NRPS-
containing gene clusters was assessed for the likelihood of it being responsible for producing
DPO, based on the structure of DPO and the gene content of each gene cluster.
The putative DPO biosynthetic cluster shared a high degree of homology to the biosynthetic
gene cluster for the pyrrole-amide, congocidine, identified in S. ambofaciens ATCC 23877T.
The putative acyl-CoA synthetase encoded by SPR_52860, the A domain encoded by
SPR_53060, the putative Cy domain encoded by SPR_53040, the TE domain encoded by
SPR_53090, the cinnamate-CoA ligase encoded by SPR_60150 and the putative D-lactate
dehydrogenase encoded by SPR_60260 were identified as genes that would be disrupted in
order to determine their involvement in DPO biosynthesis. The development of a DNA
transformation method for the introduction of exogenous DNA into S. polyantibioticus SPRT
and subsequent gene disruption experiments are described in the following chapter.
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CHAPTER 4
DEVELOPMENT OF A
TRANSFORMATION PROTOCOL
FOR STREPTOMYCES
POLYANTIBIOTICUS SPRT AND
GENE DISRUPTION EXPERIMENTS
152
CHAPTER 4
DEVELOPMENT OF A TRANSFORMATION PROTOCOL
FOR STREPTOMYCES POLYANTIBIOTICUS SPRT AND GENE
DISRUPTION EXPERIMENTS 4.1 Abstract ...................................................................................................................... 154
4.2 Introduction ................................................................................................................ 156
4.3 Materials and Methods ............................................................................................... 163
4.3.1 Bacterial strains and plasmids ........................................................................ 163
4.3.2 Media and culture conditions ......................................................................... 164
4.3.3 Primer design ................................................................................................. 170
4.3.4 PCR protocols ................................................................................................ 172
4.3.4.1 Amplification of genes for cloning into plasmid vectors....... 172
4.3.4.2 Colony PCR ........................................................................... 173
4.3.5 Cloning and restriction endonuclease digestions ........................................... 173
4.3.6 Determination of the antibiotic susceptibility of S. polyantibioticus SPRT ... 176
4.3.7 Knockout construction by gene disruption experiments ................................ 176
4.3.7.1 Conjugation ............................................................................ 176
4.3.7.1.1 Transformation of plasmid DNA into E. coli
ET12567/pUZ8002 .................................................... 176
4.3.7.1.2 Conjugation between E. coli ET12567/pUZ8002 and
E. coli JM109 ............................................................. 177
4.3.7.1.3 Intergeneric conjugation between E. coli and
S. polyantibioticus SPRT ............................................ 177
4.3.7.1.4 Confirmation of single crossover exconjugants by PCR
amplification .............................................................. 179
153
4.3.7.2 Electroporation ....................................................................... 180
4.3.7.2.1 Preparation of plasmid DNA for electroporation ....... 180
4.3.7.2.2 Electroporation ........................................................... 180
4.3.7.3 Protoplast transformation ....................................................... 183
4.3.7.3.1 Preparation of protoplasts .......................................... 183
4.3.7.3.2 Protoplast transformation ........................................... 184
4.3.8 Fermentation and isolation of DPO ............................................................... 184
4.3.9 High performance liquid chromatography (HPLC) ....................................... 186
4.3.10 Thin layer chromatography (TLC) bioautography analysis ........................... 187
4.4 Results and Discussion .............................................................................................. 188
4.4.1 Determination of the antibiotic susceptibility of S. polyantibioticus SPRT ... 188
4.4.2 The development of an optimized transformation protocol for
S. polyantibioticus SPRT ................................................................................ 188
4.4.2.1 Electroporation and protoplast transformation ...................... 188
4.4.2.2 Intergeneric conjugation ........................................................ 192
4.4.3 Gene disruption using the optimized intergeneric conjugation method ........ 196
4.4.4 Isolation of DPO from S. polyantibioticus SPRT and confirmation of its
activity against M. aurum A+ ........................................................................ 202
4.4.5 TLC bioautography analysis to determine the putative involvement of the
target genes in DPO biosynthesis .................................................................. 204
4.5 Conclusion ................................................................................................................. 210
4.6 Reference list ............................................................................................................. 211
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CHAPTER 4
DEVELOPMENT OF A TRANSFORMATION PROTOCOL
FOR STREPTOMYCES POLYANTIBIOTICUS SPRT AND GENE
DISRUPTION EXPERIMENTS
4.1 ABSTRACT
In order to identify the genes involved in DPO biosynthesis, a DNA transformation protocol
was required for the introduction of plasmid DNA into S. polyantibioticus SPRT, so that
specific genes could be disrupted to establish whether their products are involved in the
biosynthesis of DPO. Although there are several published transformation protocols for the
introduction of DNA into streptomycetes, there is no method that is generally applicable to all
species. Due to the fact that S. polyantibioticus SPRT is a novel antibiotic-producing
actinobacterium, it was necessary to develop a unique DNA transformation method.
In this study, the classical methods of electroporation and protoplast transformation of plasmid
DNA proved unsuccessful in the transformation of S. polyantibioticus SPRT. However, an
optimised method of intergeneric conjugation using the methylation-deficient E. coli
ET12567/pUZ8002 strain allowed the transfer plasmid DNA into S. polyantibioticus SPRT with
a conjugation frequency of 8.3 x 10-6 exconjugants per 1 x 107 donor E. coli cells, thereby
providing a platform for the gene disruption experiments.
Subsequently, the genes identified from the S. polyantibioticus SPRT genome sequence
(Chapter 3) as being putatively involved in the biosynthesis of DPO were individually cloned
into the suicide vector, pOJ260, transformed into S. polyantibioticus SPRT by means of mating
with E. coli ET12567/pUZ8002 and insertionally activated via homologous recombination in
the recipient strain. The genes that were disrupted were: the A domain encoded by gene
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SPR_53060, the putative Cy domain encoded by gene SPR_53040, the acyl-coA synthetase
encoded by gene SPR_52860, the cinnamate-CoA ligase encoded by gene SPR_60150, the
putative D-lactate dehydrogenase encoded by gene SPR_60250, the thioesterase encoded by
gene SPR_53090 and a selection of the A domains identified in Chapter 2.
The method for isolating DPO was carried out on each of the mutant strains, S. polyantibioticus
∆AD2, S. polyantibioticus ∆A18, S. polyantibioticus ∆A16, S. polyantibioticus ∆A28, S.
polyantibioticus ∆A7, S. polyantibioticus ∆A99, S. polyantibioticus ∆PAAK, S.
polyantibioticus ∆CYC, S. polyantibioticus ∆LAC, S. polyantibioticus ∆ACY, S.
polyantibioticus ∆CIN and S. polyantibioticus ∆THI, and the extracts were assayed for activity
against M. aurum A+. The absence of activity against M. aurum A+ in the extracts from strains
S. polyantibioticus ∆A99, S. polyantibioticus ∆CYC and S. polyantibioticus ∆ACY suggested
the involvement of these genes in the biosynthesis of DPO.
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4.2 INTRODUCTION
Streptomycetes are well known for their ability to produce commercially useful secondary
metabolites with a variety of biological activities (Hopwood, 1989). In order to discover novel
biochemical pathways used in the production of these important compounds, or to enhance or
modify their production, it is necessary to manipulate the producing strains by genetic
engineering techniques. This requires transformation of exogenous DNA into the producing
strains and subsequent gene disruption experiments (Mazy-Servais et al., 1997).
Due to the fact that streptomycetes are not naturally competent for the uptake of exogenous
plasmid DNA and because a general system for competence induced by cold shock and calcium
treatment, such as that developed for E. coli, has not yet been developed for Streptomyces, it
has hampered progress in Streptomyces strain manipulation (Marcone et al., 2010; Kieser et
al., 2000). The work by Okanishi et al. (1974) on the preparation, regeneration and transfection
of protoplasts in Streptomyces spp. led to the discovery that plasmid DNA could be transformed
into protoplasts at a very high frequency in the presence of polyethylene glycol (PEG; Bibb et
al., 1978). Even though this method, which was subsequently modified and adapted, is still
widely used in the transformation of Streptomyces species, it became apparent after limited
success in species such as Streptomyces fradiae (Matshushima and Baltz, 1985), that no method
exists which is equally efficient for all Streptomyces species and strains. Each transformation
method therefore requires the optimization of each step for each individual strain (Mazy-
Servais et al., 1997).
The reason for the different response to protoplast transformation from taxonomically related
species may be linked to the variation in the composition and density of the bacterial cell wall.
Indeed, variation in the peptidoglycan structure in response to environmental conditions, aging,
and maturation in order to adapt to changing conditions may explain the intraspecific variability
in the susceptibility to cell wall digestion (Marcone et al., 2010; Vollmer, 2008). Another
serious limitation to this method is the occurrence of restriction-modification (R-M) systems,
which are widespread in streptomycetes. R-M systems are vital components of prokaryotic
defence mechanisms against invading foreign DNA, such as phage genomes, that can decrease
transformation frequencies or make transformants undetectable, depending on the origin of the
DNA being introduced (Vasu & Nagaraja, 2013; MacNeil, 1988). The R-M systems generally
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consist of two different enzymatic activities: a restriction endonuclease (REase) and a
methyltransferase (MTase). The REase is able to recognize and cleave foreign DNA sequences
at specific sites, whereas the MTase activity ensures differentiation between self and non-self
DNA by transfer of methyl groups to specific DNA sequences within the host’s genome (Figure
4.1) (Vasu & Nagaraja, 2013). However, methyl-specific restriction systems have also been
described (Raleigh & Wilson, 1986; Lacks & Greenberg, 1977), whereby foreign methyl-
modified DNA is restricted and the host strain does not modify its own DNA. Indeed,
Streptomyces avermitilis NRRL 8165 contains a unique restriction system that restricts plasmid
DNA containing N6-methyladenine or 5-methylcytosine. Additionally, S. coelicolor A3(2) is
known to strongly restrict DNA from modification-proficient E. coli strains such as E. coli
K12, but readily accepts it when it originates from methylation-deficient strains (Kieser et al.,
2000; Flett et al., 1997; Kieser & Hopwood, 1991). Consequently, for the purpose of
transforming exogenous DNA into S. coelicolor A3(2), it is possible to thwart the R-M system
by isolating plasmid DNA from the methylation-deficient E. coli ET12567 strain (Kieser et al.,
2000). Generally, it is also possible to limit the restriction barrier by subjecting protoplasts to
heat treatment prior to transformation which serves to inactivate the restriction enzymes
(Hussain & Ritchie, 1991; Bailey & Winstanley, 1986).
Even though PEG-mediated transformation of protoplasts has allowed for the rapid
development of gene cloning systems in various Streptomyces species such as S. lividans 66,
S. rimosus R6 and S. coelicolor A3(2) amongst others, transformation of fragile protoplasts is
tedious and frequently not reproducible, and therefore many strains remain recalcitrant to
transformation (Pigac & Schrempf, 1995).
An alternative method to the use of chemicals in assisting the uptake of DNA by bacterial cells
is electroporation, which involves the application of a brief, high voltage electrical pulse to a
suspension of cells and DNA that results in the formation of transient membrane pores and
subsequent uptake of DNA by the cells (Pigac & Schrempf, 1995; Shigekawa & Dower, 1988).
Several Streptomyces species, such as Streptomyces parvulus IMET 41380 (Mazy-Servais et
al., 1997), Streptomyces vinaceus NCIB 8852 (Macy-Servais et al., 1997), S. lividans ATCC
1326 (Tyurin et al., 1995) and S. virginiae ATCC 13161 (Tyurin et al., 1995), have been
successfully transformed using electroporation. Due to the fact that electroporation avoids the
need to optimise conditions for protoplast preparation and regeneration, it is less tedious and
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time consuming than protoplast transformation and has been reported to be particularly useful
in transforming previously “untransformable” strains (Pigac & Schempf, 1995). However,
conditions for electroporation have also been described as being strain-specific (Kieser et al.,
2000).
Figure 4.1 Illustration depicting R-M systems in their role as a defence mechanism in prokaryotes. R-M
systems are able to identify the methylation status of invading foreign DNA, as methylated
sequences are recognised as “self”, while any recognition sequences that lack methylation are
identified as “non-self” and cleaved by a REase. This is due to the fact that “self” DNA is
methylated at its appropriate recognition sites by a cognate MTase belonging to the specific R-
M system (Vasu & Nagaraja, 2013).
In recent years, there has been considerable interest in the use of intergeneric conjugation,
which evades restriction barriers, as a means of transferring plasmid DNA into actinomycetes.
This method allows for the construction and manipulation of recombinant plasmids in a host
such as E. coli, which is used to transfer the genetic material into the required recipient via
horizontal gene transmission (Giebelhaus et al., 1996). Conjugal DNA transfer is a highly
conserved mechanism that is generally mediated by plasmids which encode many critical
transfer-related functions (Mazodier & Davies, 1991). The first plasmid-mediated transfer to
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be described involved the E. coli F plasmid, which has served as the model for nearly all
subsequently described conjugative plasmids from both Gram-negative and Gram-positive
bacteria (Lederberg and Tatum, 1946). Briefly, the plasmid DNA molecule is
endonucleolytically cleaved on a single strand by a plasmid-encoded relaxase protein, which
leads to the formation of a DNA-protein complex known as a relaxosome (Lanka & Wilkins,
1995). The cleaved strand is then transferred from the donor to the recipient cell via a bridge-
like structure known as a pilus (which connects the two cells). After conjugation, the donor
and recipient both contain the plasmid, which allows the recipient to serve as a donor in future
matings (Willetts & Wilkins, 1984).
In contrast to this conserved mechanism of conjugation witnessed throughout most of the
bacterial domain, conjugative plasmids of Streptomyces origin employ a different mode of
DNA transfer (Grohmann et al., 2003). The conjugative elements and transfer functions
present in Streptomyces plasmids are unique and encode fewer transfer functions compared to
plasmids from other genera. Indeed, in comparison to the approximately 25 functions required
by the E. coli F plasmid to mobilize DNA, the S. lividans plasmid pIJ101 only requires a 70
kDa membrane-associated product of the tra gene and the cis-acting locus of transfer (clt) to
mediate plasmid transfer (Grohmann et al., 2003). Moreover, the major transfer proteins of
conjugative Streptomyces plasmids are homologous to proteins which mobilize double-
stranded DNA in processes such as sporulation and cell division, instead of sharing similarity
with relaxases (Begg et al., 1995; Wu et al., 1995; Hopwood & Kieser, 1993). Additionally,
the conjugative transfer of Streptomyces plasmids has been shown to occur via a double-
stranded intermediate. Due to the fact that the Streptomyces conjugative system is unique,
there has been considerable interest in elucidating the manner in which streptomycetes mediate
plasmid transfer via conjugation (Grohmann et al., 2003; Possoz et al., 2001).
It was initially assumed that the transfer of plasmids by conjugation was confined to closely
related bacterial species or genera until Trieu-Cuot et al. (1987) dispelled this misconception
by demonstrating conjugation between Gram-negative E. coli and various Gram-positive
bacteria, which included Streptococcus lactis, Bacillus thuringiensis, S. aureus and
Enterococcus faecalis, amongst others. The shuttle plasmid used in the study, pAT187,
contained the origin of replication for E. coli and a broad-host range origin of replication for
Gram-positive bacteria. The transfer of the plasmid was contingent on the origin of transfer
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(oriT) of the incompatability group P (IncP) broad-host range E. coli plasmid RK2 acting in
cis and the transfer gene (tra) functions of the IncPα E. coli plasmid RP4 supplied in trans
(Gormley & Davies, 1991; Mazodier et al., 1989). Since then, additional studies with similarly
constructed shuttle plasmids have shown that conjugative transfer between phylogenetically
unrelated microorganisms is possible (Gormley & Davies, 1991).
The first protocol for the intergeneric transfer of plasmids between E. coli and Streptomyces
spp. was reported by Mazodier et al. (1989). The success of this transfer was dependent on the
presence of a 760 bp, cis-acting, oriT-containing fragment from the plasmid RK2 and on the
conjugative functions, such as the Tra2 core of plasmid RP4, which encodes the DNA transport
apparatus important for pilus formation, supplied in trans by the E. coli donor strain. The same
method has been used successfully with several Streptomyces species such as S. fradiae, S.
ambofaciens, S. lividans and S. coelicolor (Bierman et al., 1992) and other actinomycetes such
as Amycolatopsis (Stegmann et al., 2001), Actinoplanes (Heinzelmann et al., 2003) and
Saccharopolyspora (Matshushima et al., 1994). Importantly, the conjugative functions of
plasmid RP4 and the RK2 derivative, pUZ8002, were used to mobilize the resident plasmids
in these studies (Paranthaman & Dharmalingam, 2003). It was also demonstrated that the
major biochemical events during the intergeneric conjugal transfer occurred at the oriT, which
included the formation of a relaxosome, nicking of the closed circular dsDNA molecule and
transfer of the ssDNA intermediate from donor to recipient (Grohmann et al., 2003; Frost et
al., 1994).
Numerous cloning vectors for the conjugative transfer from E. coli to Streptomyces spp. have
been constructed, most notably by Bierman et al. (1992), who reported the construction of
plasmid and cosmid vectors that function in Streptomyces spp. due to features that allow for:
a) integration via homologous recombination between the cloned DNA fragment within the
vector and the Streptomyces chromosome (e.g. pOJ260 and pKC1132), b) autonomous
replication (e.g. pOJ446 and pKC1139) and c) site-specific integration via the bacteriophage
ΦC31 attachment site (e.g. pSET152 and pKC1163) (Kieser et al., 2000). All of these vectors
contain the 760 bp oriT fragment from plasmid RK2 and E. coli replication functions from
pUC, P15A or P1, which allow the plasmids to serve as cloning vectors for construction to
occur in an E. coli host. The recombinant plasmids can then be transferred to the desired
Streptomyces strain. Plasmids such as pOJ260 and pKC1132 were designed to be unable to
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
161
replicate in Streptomyces spp. and are therefore most useful for gene disruption experiments,
whereas plasmids such as pOJ446 and the recently designed pJN100 (Nikodinovic & Priestley,
2006), contain replication functions for Streptomyces spp. and therefore can exist as
autonomous, multicopy plasmids that are useful for complementation studies (Bierman et al.,
1992).
The use of intergeneric conjugation as a means of transforming DNA into streptomycetes has
been extensively utilized due to the simplicity of performing the method in comparison to
developing separate procedures for protoplast preparation and regeneration. Additionally,
restriction barriers such as R-M systems are by-passed or have a drastically reduced effect
when ssDNA is transferred into the recipient (Flett et al., 1997; Matshushima et al., 1994).
This is due to the fact that the majority of REases require dsDNA for the recognition and
cleavage of foreign DNA (Vasu & Nagaraja, 2013). In addition, the use of E. coli shuttle
vectors that contain oriT allow site-specific or insert-directed chromosomal integration that is
extremely useful for targeted gene disruption (Kieser et al., 2000).
In summary, electroporation and protoplast transformation methods are widely used for
introducing plasmid DNA into streptomycetes, but due to their low efficiency, intergeneric
conjugation has provided an alternative means. It has been reported that optimal conditions
for different strains may vary and therefore a defined procedure for each strain must be
established to enable a high conjugation efficiency. Furthermore, conjugation with
streptomycete mycelial fragments has proven to provide higher numbers of recombinants than
conjugation performed with freshly germinated spores (Kieser et al., 2000; Matshushima et al.,
1994).
The development of an efficient transformation method for the introduction of plasmid DNA
into a specific strain of Streptomyces allows for the targeted manipulation of biosynthetic
pathways in order to characterize individual genes and their functions. This is performed
experimentally via the disruption of target genes using the method of homologous
recombination. In this method, a DNA fragment internal to the target gene is inserted into a
plasmid vector, which integrates into the chromosome of the recipient Streptomyces strain by
a single or double homologous crossover, depending on whether a selectable marker is
contained within the DNA fragment carrying all or part of the target gene. A single crossover
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results in an integrated copy of the plasmid vector, which is flanked on either side by two
mutant alleles of the target gene i.e. one truncated at the 5' end and the other truncated at the 3'
end. A double crossover results in a mutant allele that replaces the chromosomal copy of the
target gene via two crossovers that cannot revert and is therefore more stable than a single
crossover mutant (Keiser et al., 2000).
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4.3 MATERIALS AND METHODS
4.3.1 BACTERIAL STRAINS AND PLASMIDS
All Streptomyces and E. coli strains and plasmids that were used and constructed in this study
are listed in Table 4.1. The common conjugative donor strain E. coli S17-1, which carries an
integrated derivative of IncP-group plasmid RP4 encoding all of the required plasmid
mobilization functions, was used as a donor strain for intergeneric conjugation. E. coli DH5α
was used as a general host for all standard cloning procedures. E. coli JM109 was used as a
recipient strain in conjugation with the methylation deficient E. coli ET12567 (provided by Dr
P. Whitney Swain III, Promiliad Biopharma Inc., USA). E. coli ET12567 contains the ‘driver’
plasmid pUZ8002 (derived from RK2) and was also used as the donor strain for intergeneric
conjugation. Plasmid pUZ8002 carries the plasmid RK2 transfer functions necessary for
intergeneric conjugation. Plasmid pJN100 (Figure 4.2) (provided by Dr. P. Whitney Swain III,
Promiliad Biopharma Inc., USA) (GenBank accession number: DQ309424) (Nikodinovic &
Priestley, 2006) is a high copy number E. coli-Streptomyces shuttle vector derived from
plasmid RK2 and was used for the optimization of the intergeneric conjugation method
between the different E. coli donor strains and S. polyantibioticus SPRT, as well as the
complementation experiments in S. polyantibioticus SPRT.
Plasmid pOJ260 (Figure 4.3) (provided by Dr Bohdan Ostash, Department of Genetics and
Biotechnology, Ivan Franko National University of L'viv, L'viv, Ukraine) (GenBank accession
number: GU270843) is a non-replicating, integrative E. coli-Streptomyces shuttle vector
derived from the conjugative broad host range plasmid RK2, which was used to perform gene
disruption experiments by chromosomal integration via single crossover homologous
recombination in S. polyantibioticus SPRT. Plasmid pOJ260 does not replicate in Streptomyces
and can therefore be used as a suicide vector for gene disruption and replacement (Kieser et
al., 2000).
Plasmid pJNHYG is a derivative of pJN100 carrying a hygromycin resistance cassette. This
derivative was created by first performing a restriction endonuclease digestion of pORI101
(Figure 4.4) (GenBank accession number: EF216315) (Bourn et al., 2007) using NotI to release
a 2.44 kb fragment containing the entire hygromycin resistance cassette. Concomitantly, the
plasmid pJN100 was subjected to a restriction endonuclease digestion with NotI. The 2.44 kb
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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hygromycin resistance cassette was ligated into the NotI site of the restriction endonuclease
digested pJN100 using the Rapid DNA Ligation Kit (Thermo Scientific) (14 h at 4 °C)
according to the manufacturer’s instructions to generate the 8.9 kb pJNHYG plasmid. The T4
DNA ligase was inactivated at 80 °C for 20 min before transformation into E. coli DH5α. The
plasmid was isolated using a NucleoSpin Plasmid Isolation Kit (Machery Nagel, Germany)
according to the manufacturer’s instructions.
All of the other plasmids utilized in this study are derivatives of pOJ260 and pJN100 carrying
the PCR-amplified target genes used in the gene disruption experiments (section 4.3.7). S.
polyantibioticus SPRT was isolated by Dr Paul Meyers. S. coelicolor A3(2) (NRRL B-16638)
was obtained from the culture collection of the Agricultural Research Service, United States
Department of Agriculture (NRRL) (Peoria, Illinois, USA).
4.3.2 MEDIA AND CULTURE CONDITIONS
All Streptomyces strains used in this study were grown on ISP4 agar (10 g soluble starch, 1 g
K2HPO4, 1 g MgSO4, 2 g (NH4)2SO4, 1 g CaCO3, 1 mg FeSO4, 1 mg MgCl2, 1 mg ZnSO4, 18
g bacteriological agar per litre of dH20, pH 7.2), (Shirling & Gottlieb, 1966) at 30 °C for 7 days
in order for sporulation to occur, after which the spores were scraped off the surface of the agar
using a sterile inoculating loop and stored in 20 % glycerol at -20 °C until needed. Liquid SMC
medium (10 g glucose, 4 g yeast extract, 4 g peptone, 4 g K2HPO4, 2 g KH2PO4, 0.5 g MgSO4
per litre of dH20, pH 7.0) (Zhang et al., 1992) containing 10.3 % sucrose, ISP2 (YEME)
medium (10 g malt extract, 4 g glucose, 4 g yeast extract per litre of dH20, pH 7.3) (Shirling &
Gottlieb, 1966), Luria-Bertani broth (LB) (10 g tryptone, 5 g yeast extract, 5 g NaCl, per litre
of dH20) (Sambrook et al., 1989) and V medium (2.4 g soluble starch, 10 g glucose, 3 g meat
extract, 5 g yeast extract, 5 g tryptose per litre of dH20, pH 7.2) (Marcone et al., 2010) were
used to culture S. polyantibioticus SPRT for the collection of mycelium for intergeneric
conjugation experiments. CRM (10 g glucose, 100.3 g sucrose, 10 g MgCl2.6H20, 15 g tryptic
soy broth (TSB), 5 g yeast extract per litre of dH20, pH 7.2), tryptic soy broth (TSB)
supplemented with 0.24 mM threonine and 1 % glycine and YEME supplemented with 34 %
sucrose and 0.5 % glycine were used to culture S. polyantibioticus SPRT for electroporation of
mycelia. Solid agar media used for the cultivation of S. polyantibioticus SPRT and S. coelicolor
A3(2) exconjugants were mannitol soya (MS) medium (20 g mannitol, 20 g soya flour, 20 g
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
165
bacteriological agar per litre of tap water, pH 7.0), Middlebrook 7H9, YEME and ISP4 all
containing 10 or 20 mM MgCl2. Electroporated mycelia were plated on TSB agar or R2YE
agar (103 g sucrose, 0.25 g K2SO4, 10.12 g MgCl2.6H2O, 10 g glucose, 0.1 g casamino acids,
KH2PO4, 10 ml 0.5 % KH2PO4, 80 ml 3.68 % CaCl2.2H2O, 15 ml 20 % L-proline, 100 ml 5.73
% TES buffer, 2 ml trace elements solution (40 mg ZnCl2, 200 mg FeCl3.6H2O, 10 mg
CuCl2.2H2O, 10 mg MnCl2.4H2O, 10 mg Na2B4O7.10H2O, 10 mg (NH)4Mo7O24.4H2O per litre
of dH2O), 0.5 ml 1 N NaOH, 40 ml 10 % yeast extract per litre of dH20, pH 7.2). Finally,
hypertonic V medium was used to cultivate S. polyantibioticus SPRT for preparing protoplasts,
while R2YE or VMSO.1 (2.4 g soluble starch, 0.1 g glucose, 0.3 g meat extract, 0.5 g yeast
extract, 0.5 g tryptose, 3.5 g L-proline, 4 g bacteriological agar per litre of dH20, pH 7.2) media
were used for the regeneration of protoplasts. LB broth supplemented with antibiotics (50
µg/ml apramycin, 34 µg/ml chloramphenicol and 50 µg/ml kanamycin, as required) was used
for the cultivation of all E. coli strains.
Table 4.1 Strains and plasmids used in this study
Strain/plasmid Genotype/characteristics Reference
Strains
S. polyantibioticus
SPRT Wild type This study
∆AD2 SPRT derivative carrying an integrated copy of pOJAD2; aprR This study
∆A16 SPRT derivative carrying an integrated copy of pOJA16; aprR This study
∆A18 SPRT derivative carrying an integrated copy of pOJA18; aprR This study
∆A28 SPRT derivative carrying an integrated copy of pOJA28; aprR This study
∆A7 SPRT derivative carrying an integrated copy of pOJA7; aprR This study
∆PAAK SPRT derivative carrying an integrated copy of pOJPAAK; aprR This study
∆A99 SPRT derivative carrying an integrated copy of pOJA99; aprR This study
∆CYC SPRT derivative carrying an integrated copy of pOJCYC; aprR This study
∆ACY SPRT derivative carrying an integrated copy of pOJACY; aprR This study
∆LAC SPRT derivative carrying an integrated copy of pOJLAC; aprR This study
∆CIN SPRT derivative carrying an integrated copy of pOJCIN; aprR This study
∆THI SPRT derivative carrying an integrated copy of pOJTHI; aprR This study
PJNACY ∆ACY derivative complemented with pJNACY; aprR This study
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
166
S.coelicolor
A3(2) Wild type NRRL
E. coli
ET12567 F- dam13::Tn9 dcm6 hsdM hsdR zjj202::Tn10 recF143 galK2
galT22 ara14 lacY1 xyl5 leuB6 thi1 tonA31 rpsL136 hisG4 tsx78
mtl1 glnV44
MacNeil et al.
(1992)
DH5α F- deoR endA1 recA1 relA1 gyrA96 hsdR17(rk-, mk+) supE44 thi-
1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15 λ-
Bioline (UK)
S17-1 recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 Simon et al.
(1983)
dam- dcm- F- dam-13:Tn9(CamR) dcm-6 ara-14 hisG4 leuB6 thi-1 lacY1
galK2 galT22 glnV44 hsdR2 xylA5 mtl-1 rpsL 136(StrR) rtbD1
tonA31 tsx78 mcrA mcrB1
Bioline (UK)
JM109 endA1, recA1, gyrA96, thi, hsdR17 (rk–, mk
+), relA1, supE44,
Δ(lac-proAB)
Yanisch-Perron
et al. (1985)
Plasmids
pGEM®-T Easy 3015 bp vector with 3’ - T overhangs, designed for cloning PCR
products; AmpR oriT lacZ
Promega
(USA)
pGEMAD-2 pGEM®-T Easy vector harbouring the AD2 PCR product This study
pGEMA-7 pGEM®-T Easy vector harbouring the A7 PCR product This study
pGEMA-18 pGEM®-T Easy vector harbouring the A18 PCR product This study
pGEMA-16 pGEM®-T Easy vector harbouring the A16 PCR product This study
pGEMA-28 pGEM®-T Easy vector harbouring the A28 PCR product This study
pGEM-PAAK pGEM®-T Easy vector harbouring the paaK PCR product This study
pGEMA-99 pGEM®-T Easy vector harbouring the A99 PCR product This study
pGEM-CYC pGEM®-T Easy vector harbouring the CYC PCR product This study
pGEM-ACY pGEM®-T Easy vector harbouring the ACY PCR product This study
pGEM-LAC pGEM®-T Easy vector harbouring the LAC PCR product This study
pGEM-CIN pGEM®-T Easy vector harbouring the CIN PCR product This study
pGEM-THI pGEM®-T Easy vector harbouring the THI PCR product This study
pJN100 6460 bp E. coli-Streptomyces shuttle vector derived from RK2; aprR
oriT snpA repColE1 reppIJ101
Nikodinovic &
Priestley,
(2006)
pOJ260 3469 bp non-replicating integrative E. coli-Streptomyces shuttle
vector derived from pKC787; aprR reppuc oriT lacZ
Bierman et al.
(1992)
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
167
pOJAD2 720 bp PstI/SacII fragment of pGEMAD-2 inserted into the
PstI/SacII sites of pOJ260
This study
pOJA18 670 bp PstI/SacII fragment of pGEMA-18 inserted into the
PstI/SacII sites of pOJ260
This study
pOJA16 700 bp PstI/SacII fragment of pGEMA-16 inserted into the
PstI/SacII sites of pOJ260
This study
pOJA28 708 bp PstI/SacII fragment of pGEMA-28 inserted into the
PstI/SacII sites of pOJ260
This study
pOJA7 717 bp PstI/SacII fragment of pGEMA-7 inserted into the
PstI/SacII sites of pOJ260
This study
pOJPAAK 700 bp PstI/SacII fragment of pGEM-PAAK inserted into the
PstI/SacII sites of pOJ260
This study
pOJA99 719 bp EcoRI fragment of pGEMA-99 inserted into the EcoRI site
of pOJ260
This study
pOJCYC 980 EcoRI fragment of pGEM-CYC inserted into the EcoRI site of
pOJ260
This study
pOJACY 921 bp EcoRI fragment of pGEM-ACY inserted into the EcoRI site
of pOJ260
This study
pOJLAC 608 bp PstI/SacII fragment of pGEM-LAC inserted into the
PstI/SacII sites of pOJ260
This study
pOJCIN 649 bp PstI/SacII fragment of pGEM-CIN inserted into the
PstI/SacII sites of pOJ260
This study
pOJTHI 473 bp PstI/SacII fragment of pGEM-THI inserted into the
PstI/SacII sites of pOJ260
This study
pJNACY 1557 bp EcoRI fragment of pGEM-ACY inserted into the EcoRI
site of pOJ260
This study
pUZ8002 Non-transmissable oriT-mobilizing RK2 derivative; dam dcm hsdS
Strr Telr Clmr Kmr
Paget et al.
(1999)
pJNHYG Derivative of pJN100 carrying a 2.44 kb hygromycin resistance
casette; aprR hygR oriT snpA repColE1 reppIJ101
This study
pORI101 5316 bp Mycobacterium-E. coli shuttle vector derived from
pAL5000; hygR Ori2 OriM reppMB1 reppAL5000 lacZ
Bourn et al.
(2007)
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
168
Figure 4.2 Plasmid map of the E. coli-Streptomyces shuttle expression vector, pJN100, indicating the
position of selected restriction endonuclease sites (multiple sites for the same enzyme are shown
in red).. The red arrow represents an apramycin resistance cassette, a selectable marker in both
hosts. The grey arrow represents an oriTRK2 insertion that allows intergeneric conjugation
between E. coli and Streptomyces. The orange arrow represents the origin of replication of
plasmid pIJ101, which allows for replication to take place in the Streptomyces host, while the
purple arrow represents the origin of replication of plasmid ColE1, which allows for replication
to take place in the E. coli host. The blue arrow indicates the presence of the snpR gene, the
product of which activates the snpA promoter, for use in protein expression studies. The position
of a multiple cloning region (showing only the restriction sites relevant to this study) is indicated
as MCS (Nikodinovic & Priestley, 2006).
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
169
Figure 4.3 Plasmid map of the non-replicating integrative E. coli-Streptomyces shuttle vector, pOJ260,
indicating the position of selected restriction endonuclease sites (multiple sites for the same
enzyme represented by red text). The red arrow represents an apramycin resistance cassette, a
selectable marker in both hosts. The grey arrow represents an oriTRK2 insertion that allows
intergeneric conjugation between E. coli and Streptomyces, while the orange arrow represents
the origin of replication of plasmid pUC18, which allows for replication to take place in the
E. coli host. The green arrow represents the α-peptide coding region of the E. coli lacZ gene,
encoding the enzyme β-galactosidase, which also includes restriction sites conveniently
arranged as a multiple cloning region (Bierman et al., 1992).
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
170
Figure 4.4 Plasmid map of the mycobacterial shuttle vector, pORI101. The NotI restriction sites used in a
restriction endonuclease digestion to generate a 2.44 kb fragment containing the hygromycin
resistance gene, hygA, are depicted in red text. The mycobacterial replicon genes are
represented by RepA and RepB. The mycobacterial origin of replication is represented by OriM
(Bourn et al., 2007).
4.3.3 PRIMER DESIGN
PCR primers were designed for the amplification of the following genes based on the S.
polyantibioticus SPRT genome sequence (Chapter 3): the putative Cy domain (CYC) encoded
by gene SPR_53040, the A domain (AD99) encoded by gene SPR_53060, the acyl-CoA
synthetase (ACY) encoded by gene SPR_52860, the cinnamate CoA ligase (CIN) encoded by
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
171
gene SPR_60150, the putative D-lactate dehydrogenase (LAC) encoded by gene SPR_60250
and the thioesterase (THI) encoded by gene SPR_53090. The primer sequences and their
respective amplification product sizes are shown in Table 4.2.
Additionally, the primer set, ACYORF_F/ACYORF_R, was designed to amplify the full open
reading frame of the acyl-CoA synthetase (gene SPR_52860) and incorporated the ClaI
restriction enzyme recognition sequence at the 5' end of the ACYORF_F primer sequence and
the HindIII restriction enzyme recognition sequence at the 5' end of the ACYORF_R primer
sequence, in order to achieve the in-frame cloning of this gene into plasmid pJN100.
Table 4.2 Oligonucleotide primers designed in this study
Primer Name Primer Sequence (5'→3')
Expected product size for primer
pair ADFWD GTACACCTCGGGATCCACC
719 bp ADREV TCGCCCGTGCGGTACATGC
CYCFWD CGTGGTCGACGGAGCTCC 980 bp
CYCREV GGTGTAGAAGCCGAGATCG
THIF TATGCGTGCGTTCTTCACCG 473 bp
THIR AAGGTGCATTCGAGATAGGG
CINF CCCAGTACGACCTCTCCTCC 649 bp
CINR CGGCGGATCTTCTTGTACGG
LACF ACTTCGACCTGCTGAGTACG 608 bp
LACR CAGGAACTCCGCCTTGTACG
ACYF CTGTGGGAGCTCATCGACC 921 bp
ACYR CTCGGTGCTTCCGTAGACC
ACYORF_F ATTATAATCGATGCCCTCGACCACTGAATCCG 1557 bp
ACYORF_R GATAATAAGCTTTCAGGCCGTGTCGTCCCGGTGC
POJFWD GCTGCAAGGCGATTAAGTTGG Variable
POJREV CAGGCTTTACACTTTATGCTTCC “Variable” denotes that the expected product size changes with each different target gene being amplified
due to the fact that a different target gene specific primer is used in conjunction with either POJFWD or
POJREV (section 4.3.7.1.4)
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
172
4.3.4 PCR PROTOCOLS
All PCR amplifications were performed using a Techne TC512 Thermal Cycler fitted with a
heated lid and gradient sample block.
4.3.4.1 AMPLIFICATION OF GENES FOR CLONING INTO PLASMID
VECTORS
Amplification of the A domain in gene SPR_53060 and the putative Cy domain in gene
SPR_53040 were performed using the ADFWD/ADREV and CYCFWD/CYCREV primer
sets, respectively. The cycling conditions used for the amplification of both domains were as
follows: initial denaturation at 98 oC for 1 min, followed by 35 cycles of denaturation at 98 oC
for 10 s, annealing at 55 oC for 30 s and elongation at 72 oC for 2 min, with a final elongation
at 72 oC for 10 min. PCR reactions consisted of: 100 ng of DNA, 1 U Phusion High-Fidelity
DNA polymerase (Thermo Fisher Scientific, USA), 0.5 μM of each primer, 0.2 mM of each
dNTP, 1 x Phusion HF buffer and 4 % (v/v) glycerol in a total volume of 50 μl.
Amplification of sections of the cinnamate CoA ligase (gene SPR_60150), the putative D-
lactate dehydrogenase (gene SPR_60250), the thioesterase (gene SPR_53090) and the acyl-
CoA synthetase (gene SPR_52860) were performed using the CINF/CINR, LACF/LACR,
THIF/THIR, ACYF/ACYR and ACYORF_F/ACYORF_R primer sets, respectively. The
cycling conditions used for the amplification of these genes were similar to those used for the
amplification with the ADFWD/ADREV and CYCFWD/CYCREV primer sets, except that the
annealing temperature was 51 oC for the THIF/THIR primer set, 53 oC for the ACYF/ACYR
primer set, 56 oC for the CINF/CINR and LACF/LACR primer sets and 60 oC for the
ACYORF_F/ACYORF_R primer set. PCR reactions were set up as described for the
amplification of the A and Cy domains mentioned above, with the exception of the glycerol
concentration, which was 2 % (v/v) for amplification using the CINF/CINR, LACF/LACR,
THIF/THIR and ACYF/ACYR primer sets and 8 % (v/v) for the ACYORF_F/ACYORF_R
primer set.
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
173
All PCR amplification products were resolved by electrophoresis alongside a λ-PstI molecular
marker on 0.8 % agarose gels containing 0.8 μg/ml ethidium bromide in order to analyse
amplicon size and assess primer specificity. The products were visualized using a long
wavelength UV light box (Bio-Rad Gel Doc EQ-system™, Bio-Rad Laboratories Inc., USA).
4.3.4.2 COLONY PCR
A colony PCR protocol was used to confirm the presence and correct size of the amplification
product in transformants harbouring recombinant constructs. The PCR cycling conditions for
the amplification of these inserts was as follows: initial denaturation at 95 oC for 5 min,
followed by 35 cycles of denaturation at 95 oC for 30 s, annealing at 60 oC for 90 s and
elongation at 72 oC for 60 s, with a final elongation at 72 oC for 10 min. PCR reactions consisted
of: a toothpick-tip size amount of cell mass from a transformant colony, 2 U SuperThem Taq
polymerase (JMR Holdings, USA), 0.5 μM of each primer, M13F and M13R (Table 2.1), 0.8
mM of each dNTP and 3 mM MgCl2 in a total volume of 20 μl.
All PCR amplification products were resolved by agarose gel electrophoresis alongside a λ-PstI
molecular marker on 0.8 % agarose gels, containing 0.8 μg/ml ethidium bromide, in order to
analyse amplicon size and primer specificity. The products were visualized using a long
wavelength UV light box (Bio-Rad Gel Doc EQ-system™, Bio-Rad Laboratories Inc., USA).
The fragments of interest were excised from the gel and purified using the FavorPrep Gel/PCR
Purification kit (FavorGenTM, Germany), according to the manufacturer’s instructions, if they
were to be cloned into pGEM®-T Easy for DNA sequencing (section 2.3.6), or the amplified
products were purified using the MSB® Spin PCRapace kit (STRATEC Molecular, Germany)
if they were to be sent directly for sequencing as PCR products.
4.3.5 CLONING AND RESTRICTION ENDONUCLEASE DIGESTIONS
For cloning, the fragments of interest were excised from the gel (section 4.3.4.2) and purified
using the FavorPrep Gel/PCR Purification kit (FavorGenTM, Germany) according to the
manufacturer’s instructions. The amplification products obtained from the primer sets
ADFWD/ADREV, CYCFWD/CYCREV, THIF/THIR, CINF/CINR, LACF/LACR and
ACYF/ACYR were ligated individually into the pGEM®-T plasmid as described in the
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
174
pGEM®-T Easy Vector System kit (Promega, USA). The ligation reaction was incubated at
22 °C for 14 h after which 10 ng of the reaction was transformed into E. coli α-Select Bronze
Efficiency Competent Cells (Bioline, UK) according to the manufacturer’s instructions.
The transformants were inoculated onto LB agar (Sambrook et al., 1989) supplemented with
100 µg/ml ampicillin, 40 µg/ml X-gal and 0.2 mM IPTG and incubated at 37 °C for 18 h.
Transformants harbouring recombinant pGEM®-T constructs were identified by blue/white
selection and white colonies were subcultured onto fresh LB agar plates supplemented with
100 µg/ml ampicillin, 40 µg/ml X-gal and 0.2 mM IPTG and incubated at 37 °C for 18 h to
confirm the white phenotype. The presence of the desired inserts was determined by
performing colony PCR using the M13F and M13R primer set (Table 2.1) in order to ensure
the correct size of the cloned fragment (Section 4.3.4.2).
Transformants identified as harbouring the correct recombinant constructs were inoculated into
5 ml LB broth containing 100 µg/ml ampicillin and cultured overnight with gentle shaking at
37 °C. Thereafter, plasmid DNA was isolated using the NucleoSpin Plasmid Isolation kit
(Machery Nagel, Germany) according to the manufacturer’s instructions and quantitated
spectrophotometrically as described before (section 2.3.2). One microgram of each of the
recombinant pGEM®-T vectors, individually carrying the THIF/THIR, LACF/LACR and
CINF/CINR amplification products, was subjected to a double restriction endonuclease
digestion with PstI and SacII (1.5 U of each enzyme) in the appropriate restriction buffer
overnight at 37 °C. The recombinant pGEM®-T vectors, individually carrying the
ADFWD/ADREV, CYCFWD/CYCREV and ACYF/ACYR amplification products, were
subjected to a single restriction endonuclease digestion with 1.5 U EcoRI in the appropriate
restriction buffer overnight at 37 °C. Each reaction was subjected to agarose gel
electrophoresis as described earlier and the desired inserts obtained from the restriction
endonuclease digestions were excised from the gel and purified using the Favorgen Gel/PCR
Purification Kit (FavorgenTM, Germany). The purified inserts were ligated into the plasmid
vector, pOJ260, which had been digested overnight at 37 oC with 1.5 U of EcoRI or a double
digestion consisting of SacII and PstI and thereafter dephosphorylated using rAPid Alkaline
Phosphatase (Roche, Switzerland) according to the manufacturer’s instructions. The alkaline
phosphatase incubation period was 24 h at 37 oC instead of the recommended 1 h.
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
175
Subsequently, ligation of each of the digested purified inserts into the dephosphorylated
pOJ260 vector was performed according to either the sticky-end or blunt-end protocol for the
Rapid DNA Ligation Kit (Thermo Fisher Scientific, USA) for 14 h at 4 oC, after which 10 ng
of the reaction was transformed into chemically competent E. coli α-Select Bronze Efficiency
Competent Cells (Bioline, UK) according to the manufacturer’s instructions. The
transformants were inoculated onto LB agar supplemented with 50 µg/ml apramycin, 40 µg/ml
X-gal and 0.2 mM IPTG and incubated at 37 °C for 18 h. Transformants harbouring
recombinant pOJ260 constructs were identified by blue/white selection and white colonies
were subcultured onto fresh LB plates supplemented with 50 µg/ml apramycin, 40 µg/ml X-
gal and 0.2 mM IPTG and incubated at 37 °C for 18 h to confirm the white phenotype.
Transformants with a white phenotype were identified as harbouring the correct recombinant
constructs and were inoculated into 5 ml LB broth containing 50 µg/ml apramycin and cultured
overnight with shaking at 37 °C. Thereafter, plasmid DNA was isolated using the NucleoSpin
Plasmid Isolation kit (Machery Nagel, Germany) according to the manufacturer’s instructions
and quantitated spectrophotometrically as described before (section 2.3.2). The recombinant
constructs carrying the amplification products obtained from the THIF/THIR, CINF/CINR,
LACF/LACR, ACYF/ACYR, CYCFWD/CYCREV and ADFWD/ADREV primer sets were
designated pOJTHI, pOJCIN, pOJLAC, pOJACY, pOJCYC and pOJA99, respectively (Table
4.1) and sequenced by the dideoxy chain-termination method (Sanger et al., 1977) on an
Applied Biosystems Big Dye terminator v3.1 DNA sequencer using BIOLINE Half Dye Mix
(Macrogen Inc., South Korea) using the appropriate gene specific primers in order to confirm
the presence of the desired inserts.
Additionally, the pGEMA-16, pGEMA-18, pGEMA-28, pGEMA-7 and pGEMAD-2 A
domain clones and the pGEM-PAAK clone described in Chapter 2 (section 2.3.8.2) were
subjected to restriction endonuclease digestion with 1.5 U EcoRI and the appropriate restriction
buffer at 37 °C overnight. The DNA samples were subjected to agarose gel electrophoresis,
excised from the gel and purified as described above. Each of the inserts was ligated into
dephosphorylated pOJ260, which had been subjected to restriction endonuclease digestion with
EcoRI, and transformed into chemically competent E. coli α-Select Bronze Efficiency
Competent Cells (Bioline, UK) according to the manufacturer’s instructions. Selection of
positive transformants carrying the desired insert and isolation of plasmid DNA was performed
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
176
as described above and the recombinant constructs carrying the A16, A18, A28, A7, AD2 and
paaK insert sequences were designated pOJA16, pOJA18, pOJA28, pOJA7, pOJAD2 and
pOJPAAK.
Furthermore, the amplification product obtained from the ACYORF_F/ACYORF_R primer
set was ligated directly into the pJN100 plasmid, which had been digested with 1.5 U of each
of ClaI and HindIII in the appropriate restriction buffer overnight at 37 °C. The ligation was
performed at 4 °C for 18 h and thereafter 10 ng of the ligation product was transformed into
chemically competent E. coli α-Select Bronze Efficiency Competent Cells (Bioline, UK)
according to the manufacturer’s instructions. The transformants were inoculated onto LB agar
supplemented with 50 µg/ml apramycin, 40 µg/ml X-gal and 0.2 mM IPTG and incubated at
37 °C for 18 h. Transformants harbouring recombinant pJN100 constructs were identified by
colony PCR using the ACYF/ACYR primer set (section 4.3.4.2).
4.3.6 DETERMINATION OF THE ANTIBIOTIC SUSCEPTIBILITY OF
S. POLYANTIBIOTICUS SPRT
The antibiotic susceptibility of S. polyantibioticus SPRT was determined by inoculation onto
YEME agar containing 0, 10, 15, 20, 25, 30, 35, 40, 45 and 50 µg/ml of apramycin, hygromycin
B or kanamycin and incubating for 72 h at 30 °C. Subsequently, growth of S. polyantibioticus
SPRT was assessed to determine the susceptibility to each antibiotic.
4.3.7 KNOCKOUT CONSTRUCTION BY GENE DISRUPTION EXPERIMENTS
4.3.7.1 CONJUGATION
4.3.7.1.1 TRANSFORMATION OF PLASMID DNA INTO E. COLI
ET12567/pUZ8002
Chemically competent E. coli ET12567/pUZ8002 cells were prepared according to the method
described by Dagert and Ehrlich (1979). The plasmids designated pOJA16, pOJA18, pOJA28,
pOJA7, pOJAD2, pOJPAAK, pOJTHI, pOJCIN, pOJLAC, pOJACY, pOJCYC, pOJA99 and
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
177
pJNACY were transformed into chemically competent E. coli ET12567/pUZ8002 cells for
subsequent intergeneric conjugation.
4.3.7.1.2 CONJUGATION BETWEEN E. COLI ET12567/pUZ8002
AND E. COLI JM109
In order to test the conjugal ability of E. coli ET12567/pUZ8002, conjugation between E. coli
ET12567/pUZ8002 and E. coli JM109 was performed according to the method described by
Zhang et al. (2013) with some modifications. Briefly, the donor strain, E. coli
ET12567/pUZ8002 harbouring the plasmid pOJAD2, was cultured in LB broth containing 50
µg/ml apramycin, 100 µg/ml chloramphenicol and 25 µg/ml kanamycin, while the recipient
strain, E. coli JM109, was cultured in LB broth without antibiotics at 37 °C with shaking at
200 rpm until an OD600nm = 0.5 was reached (mid-exponential phase). The cells were collected
by centrifugation at 5000 rpm for 5 min, washed once with an equal volume of LB broth
(without antibiotics) and resuspended in 1 ml LB medium before 50 µl of the donor strain was
mixed with 50 µl of the recipient in a 1.5 ml microcentrifuge tube and supplemented with 900
µl LB broth which had been pre-warmed to 37 °C. After mixing, the cells were incubated at
37 °C without shaking for 24 h. The conjugation process was interrupted by vortexing for 10
sec and the mixture was immediately serially diluted 10-fold and plated on LB agar containing
50 µg/ml nalidixic acid and 50 µg/ml apramycin in order to select for the recipient strain (strain
JM109 has a DNA gyrase mutation which makes it resistant to nalidixic acid).
4.3.7.1.3 INTERGENERIC CONJUGATION BETWEEN E. COLI
AND S. POLYANTIBIOTICUS SPRT
Classical intergeneric conjugation was performed between E. coli ET12567/pUZ8002 and
S. polyantibioticus SPRT and between E. coli ET12567/pUZ8002 and S. coelicolor A3(2),
according to the method described by Flett et al. (1997). Conjugation was also performed
between E. coli S17-1 and both Streptomyces strains according to the method described by
Mazodier et al. (1989). Briefly for both methods, the donor E. coli strain (ET12567/pUZ8002
or S17-1 carrying pJN100) was cultured in 25 ml LB broth supplemented with 50 µg/ml
apramycin, 34 µg/ml chloramphenicol and 50 µg/ml kanamycin to an OD600nm = 0.5, pelleted
by centrifugation at 5000 rpm for 5 min, washed twice with LB broth and resuspended in 1 ml
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
178
LB. Aliquots of S. polyantibioticus SPRT and S. coelicolor A3(2) spore suspensions, which
had been stored at -20 °C, were used as recipients. Spores were induced to germinate by heat-
shock at 50 °C for 10, 20, 30 or 60 min, after which the donor E. coli cells were added to the
prepared spores and the mixture was inoculated onto MS + 20 mM MgCl2 plates. The
conjugation plates were incubated for 16 h at 30 °C, after which the surface of each plate was
overlaid with 1 ml of sterile dH20 containing 500 µg nalidixic acid and 1 mg apramycin. The
plates were incubated for a further 96 h at 30 °C and the exconjugant colonies were counted.
The classical conjugation method was modified in order to obtain S. polyantibioticus SPRT
exconjugants by growing the donor E. coli culture to an OD600nm of 0.3, 0.4 or 0.6 (instead of
0.5, by decreasing the centrifugation speed to 3500 rpm (instead of 5000 rpm), by using a
culture of S. polyantibioticus SPRT which had been grown for 5 h, 12 h, 18 h, 24 h, 48 h and
72 h (instead of using a spore preparation), by cultivating S. polyantibioticus SPRT mycelia in
YEME, V or SMC media, and by plating the conjugation mixture on 7H9, YEME or ISP4
supplemented with 10 mM or 20 mM MgCl2 (instead of MS supplemented with 10 mM or 20
mM MgCl2). All of these modifications were tested as individual changes to the original
method and in different combinations in order to optimise the method for transformation of S.
polyantibioticus SPRT.
A reproducible method for intergeneric conjugation between E. coli ET12567/pUZ8002 and S.
polyantibioticus SPRT was finally established based on the methods described by Marcone et
al. (2010) and Du et al. (2012). Briefly, a culture of the donor strain, E. coli ET12567/pUZ8002
containing the selected plasmid was cultivated in 25 ml LB broth supplemented with 50 µg/ml
apramycin, 34 µg/ml chloramphenicol and 50 µg/ml kanamycin to an OD600nm = 0.4
(approximately 1 x 107 cells/ml). Cells were collected by centrifugation at 3500 rpm for 7 min,
washed twice with an equal volume of LB broth and resuspended in 1 ml LB broth. S.
polyantibioticus SPRT mycelia were prepared as follows: fresh spores were scraped off the
surface of an ISP4 plate and used to inoculate 25 ml SMC medium, containing 10.3 % sucrose
and incubated at 30 °C on a rotary shaker at 200 rpm for 18 h (exponential phase). Mycelia
were collected by centrifugation at 10 000 rpm for 10 min at 4 °C and resuspended in 2 ml ice
cold LB. For conjugation, 0.5 ml donor E. coli ET12567/pUZ8002 cells carrying the selected
plasmid (OD600nm = 0.4) were added to 0.5 ml S. polyantibioticus mycelium and incubated in a
2 ml microcentrifuge tube at 30 °C for 1 h before serially diluting 1000 fold in LB broth and
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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plating on YEME agar supplemented with 10 mM or 20 mM MgCl2. The conjugation plates
were incubated at 30 °C for 18 h and then overlaid with 25 µg/ml nalidixic acid and 25 µg/ml
apramycin. The plates were incubated for a further 72-96 h at 30 °C before single Streptomyces
colonies were sub-inoculated onto fresh YEME plates containing 50 µg/ml nalidixic acid and
30 µg/ml apramycin to confirm the single crossover exconjugants. The conjugation frequency
was calculated as the number of exconjugants per total number of donor E. coli cells.
To confirm the chromosomal integration of the pOJ260 and pJN100 derivative plasmids,
gDNA was extracted from the exconjugants using the method described in section 2.3.2 and
PCR amplification using the pOJFWD/pOJREV primers was performed (section 4.3.7.1.4).
4.3.7.1.4 CONFIRMATION OF SINGLE CROSSOVER EXCONJUGANTS
BY PCR AMPLIFICATION
Amplification of gDNA isolated from S. polyantibioticus mutant strains ∆AD2, ∆A16, ∆A18,
∆A28, ∆A7, ∆PAAK, ∆THI, ∆CIN, ∆LAC, ∆ACY, ∆CYC and ∆A99 was performed using the
POJFWD primer in combination with the respective reverse gene specific primer of interest:
A7R for AD2, A16, A18, A28 and A7, PAAKREV for PAAK, THIR for THI, CINR for CIN,
LACR for LAC, ACYR for ACY, CYCREV for CYC and ADREV for A99, in order to confirm
the integration of the target gene within the mutant S. polyantibioticus genomes. Additionally,
the POJREV primer was used in combination with the respective forward gene specific primer
of interest: A3F for AD2, A16, A18, A28 and A7, PAAKFWD for PAAK, THIF for THI, CINF
for CIN, LACF for LAC, ACYF for ACY, CYCFWD for CYC and ADFWD for A99, in an
amplification reaction to confirm the integration of the target within the mutant S.
polyantibioticus genomes.
The cycling conditions used for the amplification reactions were performed as follows: initial
denaturation at 95 oC for 5 min, followed by 35 cycles of denaturation at 95 oC for 30 s,
annealing at 56 oC for 30 s and elongation at 72 oC for 60 s, with a final elongation at 72 oC for
5 min. PCR reactions consisted of: 200 ng of DNA, 2 U SuperThem Taq polymerase (JMR
Holdings, USA), 1.5 μM of each primer, 0.2 mM of each dNTP and 3 mM MgCl2 in a total
volume of 50 μl.
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All PCR amplification products were resolved by electrophoresis alongside a λ-PstI molecular
marker on 0.8 % agarose gels containing 0.8 μg/ml ethidium bromide in order to analyse
amplicon size. The products were visualized using a long wavelength UV light box (Bio-Rad
Gel Doc EQ-system™, Bio-Rad Laboratories Inc, USA) and excised from the agarose gel,
purified using the Favorgen Gel/PCR Purification Kit (FavorgenTM, Germany) and sequenced
by the dideoxy chain-termination method (Sanger et al., 1977) on an Applied Biosystems Big
Dye terminator v3.1 DNA sequencer using BIOLINE Half Dye Mix (Macrogen Inc., South
Korea).
4.3.7.2 ELECTROPORATION
4.3.7.2.1 PREPARATION OF PLASMID DNA FOR
ELECTROPORATION
The plasmids, pJN100 and pJNHYG, were transformed into E. coli dam-/dcm- chemically
competent cells, according to the method described by Dagert and Ehrlich, (1979), in order to
generate unmethylated plasmid DNA, which was deemed necessary for efficient
electroporation (Spath et al., 2012). Thereafter, plasmid DNA was isolated using the
NucleoSpin Plasmid Isolation kit (Machery Nagel, Germany) according to the manufacturer’s
instructions and quantitated spectrophotometrically as described before (section 2.3.2).
4.3.7.2.2 ELECTROPORATION
Electroporation of plasmid DNA into S. polyantibioticus SPRT and S. coelicolor A3(2) was
initially performed based on the method described by Pigac & Schrempf (1995). Briefly, S.
polyantibioticus SPRT and S. coelicolor A3(2) mycelia was individually cultured in 25 ml CRM
at 30 °C on a rotary shaker at 220 rpm for 24-72 h. Each culture was harvested by
centrifugation at 10 000 rpm at 4 °C, washed once with 25 ml ice cold 10 % sucrose, centrifuged
at 10 000 rpm at 4 °C, re-suspended in 12.5 ml 15 % glycerol, centrifuged at 10 000 rpm at 4
°C and finally re-suspended in 2 ml ice cold 15 % glycerol containing 100 µg/ml lysozyme.
The mycelial suspension was incubated at 37 °C for 1 h and then washed twice with ice-cold
15 % glycerol by centrifugation at 10 000 rpm at 4 °C, before re-suspension in 1 ml of the
electroporation buffer, which consisted of sterile de-ionized dH20 containing 30 % PEG-1000,
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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10 % glycerol and 6.5 % sucrose. The mycelial suspension was dispensed into 50 µl aliquots
in 1.5 ml microcentrifuge tubes and immediately stored at -80 °C overnight. Subsequently, a
50 µl aliquot was thawed on ice and 10 ng to 1 µg of plasmid DNA (pJN100 or pJNHYG) was
added to it, before the mixture was transferred into a 2 mm-gapped electrocuvette (Bio-Rad
Laboratories Inc., USA) and subjected to an electrical pulse ranging from 0 to 12.5 kV/cm
using a Gene Pulser (Bio-Rad Laboratories Inc., USA), which was connected to a pulse
controller (25 µF capacitor) with a parallel resistor setting of 200, 400, 600 or 800 Ω. The
pulsed mycelium was immediately diluted with ice cold 0.75 ml CRM and incubated on a
rotary shaker at 220 rpm for 3 h at 30 °C. Before plating, CRM was added to a final volume
of 1 ml and dilutions were inoculated onto TSB plates containing 30 µg/ml apramycin. Control
reactions were performed by omitting either the plasmid DNA or the electrical pulse.
An alternative method described by Tyurin et al. (1995) for electroporation of freshly
germinated mycelial fragments was also performed. Briefly, freshly harvested
S. polyantibioticus SPRT and S. coelicolor A3(2) spores were inoculated individually into 25
ml TSB and incubated on a rotary shaker (220 rpm) at 30 °C for 3-48 h. The cells were
harvested by centrifugation at 10 000 rpm (4 °C) for 10 min, followed by washing twice with
0.15 M sucrose (centrifugation at 10 000 rpm for 10 min each time) and resuspension of the
pellet in the electroporation buffer consisting of sterile de-ionized dH20 containing 7 mM
HEPES, 75 mM sucrose and 1 mM MgCl2. The mycelial suspension was dispensed into 50 µl
aliquots and stored at -80°C overnight. Subsequently, 10 ng to 1 µg of plasmid DNA was
added to a thawed 50 µl mycelial aliquot and pulsed as described above. The pulsed mycelia
were immediately diluted with 0.5 ml TSB, left on ice for 10 min and incubated at 30 °C for
1.5 h. Each electroporation reaction was inoculated onto TSB plates containing 30 µg/ml
apramycin. Controls were performed by omitting either the plasmid DNA or the electrical
pulse.
Another two methods described by Mazy-Servais et al. (1997) and Ma et al. (2013) were also
tested. For the method described by Mazy-Servais et al. (1997), freshly harvested S.
polyantibioticus SPRT spores were inoculated into 25 ml YEME medium supplemented with
34 % sucrose and 0.5 % glycine and incubated on a rotary shaker (220 rpm) at 30 °C for 12-72
h. The mycelia were harvested by centrifugation at 10 000 rpm (4 °C) for 15 min, followed by
washing three times with an equal volume of sterile dH20 (centrifugation at 10 000 rpm for 10
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
182
min each time). Two different treatments were then applied to the cells: lysozyme digestion
or suspension in PEG-supplemented electroporation buffer. In the case of the lysozyme
treatment, cells were resuspended in 25 ml electroporation buffer (10 % sucrose, 15 % glycerol
and 3 mM NaH2PO4-Na2HPO4 buffer, pH 7.4) containing 0.5 mg/ml lysozyme and incubated
at 37 °C for 1 h, before the cells were harvested by centrifugation at 10 000 rpm (4 °C) for 15
min. The cells were resuspended in 1 ml ice cold electroporation buffer and distributed into
50 µl aliquots before electroporation. In the case of PEG treatment, cells were resuspended in
25 ml ice cold electroporation buffer, subjected to the same centrifugation conditions
mentioned above and resuspended in 1 ml electroporation buffer containing 28.5 % PEG-1000,
before distribution into 50 µl aliquots. Ten nanograms (10 ng) to one microgram (1 µg) of
plasmid DNA was then added to both sets of treated aliquots, which were kept on ice for 1 min
before the mixture was transferred to an electrocuvette and a single electrical pulse was applied
according to the conditions shown in Table 4.3. The pulsed mycelia were diluted with ice cold
0.4 ml SOC solution, kept on ice for 5 min and plated onto R2YE agar plates containing 30
µg/ml apramycin.
Table 4.3 Electroporation conditions used in this study Parallel resistance (Ω) Pulse (kV/cm) Capacitance (µF)
200 7.5 25
200 10 25
200 12.5 25
400 5 25
400 7.5 25
400 10 25
400 12.5 25
600 5 25
600 7.5 25
600 10 25
600 12.5 25
800 2.5 25
800 5 25
800 7.5 25
800 10 25
800 12.5 25
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The method described by Ma et al. (2013) was performed in a similar manner to the method
described by Tyurin et al. (1995) with some modifications. Briefly, the resuspension step
consisted of resuspending the cell pellet in 15 % glycerol containing 25 mg/ml lysozyme and
10 µg/ml penicillin G, incubating at 37 °C overnight and then for a further 2 h at room
temperature. Furthermore, the electroporation buffer consisted of 10 % PEG-1000, 10 %
glycerol and 6.5 % sucrose, each electroporated sample was diluted in 1 ml CP medium (1 %
glucose, 0.2 % tryptone, 0.4 % yeast extract, 0.05 % MgSO4.7H2O, 0.05 % K2HPO4, 0.05 %
NaCl per litre of dH2O, pH 7.2) instead of CRM and was plated on R2YE and YEME solid
media. In addition, modifications to the above mentioned methods were performed by replacing the
wash buffers with 1 M sorbitol supplemented with 2, 4, 6 or 8 % DMSO, as well as with 1 mM
citrate supplemented with 16 % raftilose and replacing the initial culture medium with TSB
supplemented with 0.24 mM threonine and 1 % glycine. Other modifications to the
electroporation conditions included double pulsing each sample and the addition of 5 µg of
TypeOne Restriction Inhibitor (Epicentre, USA) to each electroporation reaction before
pulsing in order to circumvent potential R-M systems.
4.3.7.3 PROTOPLAST TRANSFORMATION
4.3.7.3.1 PREPARATION OF PROTOPLASTS
S. polyantibioticus SPRT and S. coelicolor A3(2) protoplasts were prepared according to the
method described by Marcone et al. (2010) with some modifications. Briefly, a glycerol spore
preparation (Kieser et al., 2000) of each strain was inoculated into 25 ml V medium and
cultured for 36-48 h at 30 °C on a rotary shaker (220 rpm). The culture was centrifuged at
4500 rpm for 10 min at 4 °C and washed once (centrifugation at 3250 rpm for 10 min) with 25
ml P medium (103 g sucrose, 0.25 g K2SO4, 2.02 g MgCl2.6H2O, 2 ml trace element solution,
1 ml 0.5 % KH2PO4, 3.68 % CaCl2.2H2O, 10 ml 5.73 % TES buffer per litre of dH2O, pH 7.2)
(Okanishi et al., 1974). In order to achieve cell-wall digestion, 12.5 ml P medium (consisting
of 20 mg/ml lysozyme, 0.018 mg/ml mutanolysin and 100 mg/l pluronic) was added to the cell
pellet and incubated at 30 °C on a rotary shaker (50 rpm) for 48 h. Protoplast formation was
monitored by using an Olympus CH20 microscope at 1000 x magnification. Protoplasts were
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
184
detached from residual mycelium clumps by gently drawing through a 5 ml glass pipette tip
and then filtering through sterile cotton wool. Protoplasts were harvested by centrifugation at
4500 rpm for 10 min at 4 °C and finally resuspended in 1 ml P medium. Fifty microlitre (50
µl) aliquots of the protoplast suspension were added to microcentrifuge tubes, which were
placed in a beaker of ice and then stored at -80 °C until needed.
4.3.7.3.2 PROTOPLAST TRANSFORMATION
One microgram of plasmid DNA (approximately 5 µl) was added to each protoplast suspension
(section 4.3.7.3.1) in a microcentrifuge tube, followed by the addition of 200 µl T buffer and
gentle mixing by pipetting up and down. After incubation at room temperature for 2 min, the
transformation mixture was inoculated onto R2YE medium (Kieser et al., 2000) or VMSO.1
agar medium (Marcone et al., 2010). The plates were incubated at 30 °C for 20 h and then
overlaid with 30 µg/ml apramycin.
4.3.8 FERMENTATION AND ISOLATION OF DPO
Spores and mycelial mass were collected from S. polyantibioticus strains SPRT, ∆AD2, ∆A16,
∆A18, ∆A28, ∆A7, ∆PAAK, ∆CIN, ∆LAC, ∆CYC, ∆A99, ∆ACY, ∆THI and PJNACY from
10 day old ISP4 agar plates and heavy suspensions were made in 2 ml microcentrifuge tubes
containing 1.5 ml sterile distilled water. The spore suspension of each strain was used to
inoculate a seed culture of 25 ml Hacène’s medium (HM; 5 g glucose, 4 g yeast extract powder,
10 g malt extract powder and 1 g NaCl per litre of dH2O, pH 7.0) (Hacène & Lefebvre, 1995)
in a 250 ml Erlenmeyer flask. The flask was incubated at 30 °C for 48 h on a rotary shaker,
before inoculation into a 5 L Erlenmeyer flask containing 1 litre of HM. The flask was
incubated at 30 °C on a rotary shaker for an additional 192 h (8 days) for a total fermentation
time of 10 days.
The purity of each culture was confirmed by performing a standard Gram stain, after which
DPO was isolated and purified from the cultures according to the method depicted in Figure
4.5 (method adapted from le Roes, 2005). Briefly, the culture was filtered through a 1 x 4 sized
coffee filter (House of Coffees, RSA), after which DPO was extracted from the mycelial mass
with methanol and from the culture filtrate with ethyl acetate by stirring at room temperature
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
185
on a magnetic stirrer overnight. After concentration to 25 ml, by evaporation in a fumehood,
the methanol and ethyl acetate extracts were combined, the pH was adjusted to 7.0 (monitored
with pH strips, Merck, Germany) and the mixture was re-extracted with 500 ml toluene by
stirring at room temperature on a magnetic stirrer overnight. The toluene extract was
concentrated to 50 ml by evaporation in a fumehood and extracted with 0.1 M sodium acetate
buffer (pH 3.5) by stirring at room temperature on a magnetic stirrer overnight. The toluene
layer was concentrated to 5 ml by evaporation in a fumehood and used directly in subsequent
HPLC and TLC bioautography analysis experiments.
In addition, following the isolation of pure DPO from strains S. polyantibioticus SPRT,
S. polyantibioticus ∆AD2, S. polyantibioticus ∆A18, S. polyantibioticus ∆A16,
S. polyantibioticus ∆A28, S. polyantibioticus ∆A7, S. polyantibioticus ∆PAAK,
S. polyantibioticus ∆CYC, S. polyantibioticus ∆A99, S. polyantibioticus ∆ACY,
S. polyantibioticus ∆LAC, S. polyantibioticus ∆CIN and S. polyantibioticus ∆THI, each DPO
sample was subjected to UV spectrophotometry at a wavelength of 280 nm using the
Nanodrop® ND-1000 Spectrophotometer (Coleman Technologies Inc., USA).
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
186
Figure 4.5 Schematic diagram depicting the isolation and purification of DPO.
4.3.9 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Analytical reverse phase-HPLC analysis of a pure DPO sample (approximately 1 µg/ml)
isolated from S. polyantibioticus SPRT (section 4.3.8) and a commercial sample of DPO
(Sigma-Aldrich, USA, catalogue number: D210404) (1 mg/ml dissolved in toluene) was
carried out on an Agilent system (Agilent Technologies, USA) using a Vydac-C18 column (5
µm particle size, 4.66 x 250 mm) with 50 % acetonitrile as the mobile phase. The flow rate was
1 ml/min and DPO detection was at 280 nm.
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4.3.10 THIN LAYER CHROMATOGRAPHY (TLC) BIOAUTOGRAPHY ANALYSIS
Each pure DPO sample (approximately 1 mg/ml) (section 4.3.8), a commercial DPO sample
(Sigma-Aldrich, USA) (1 mg/ml dissolved in toluene) and the solvent, toluene, to serve as a
negative control, was applied to the surface of a silica gel 60 F254 TLC plate (Merck, Germany,
catalogue number: 1055530001) and allowed to dry in a fumehood for 15 min at room
temperature. The plates were developed in a pre-saturated solvent chamber using ethyl acetate
as the mobile phase until the solvent front reached approximately 1 cm from the top of the
plates. The developed TLC plates were removed from the chamber and allowed to air-dry for
30 min, before being subjected to bioautography analysis against M. aurum A+.
M. aurum A+ was inoculated in LB broth and incubated at 37 °C with shaking for two days.
After performing a standard Gram stain to confirm the purity of the culture, the optical density
of the culture was determined on a Novaspec II spectrophotometer (Pharmacia Biotech,
Sweden) at a wavelength of 600 nm. The culture was diluted to OD600nm = 0.5 using sterile LB
broth. The test bacterium was applied to the surface of the TLC plate (containing the DPO test
sample, commercial DPO sample and toluene) with sterile non-absorbent cotton wool, the TLC
plate was placed in a plastic sealable container lined with moist paper towel and incubated
overnight at 37 °C (about 18 h).
Thiazolyl blue (MTT; Sigma-Aldrich, USA, catalogue number: M2128), dissolved in
phosphate buffered saline (4.26 g Na2HPO4.7H2O, 2.27 g KH2PO4, 8 g NaCl per litre of dH2O,
pH 7.0) at a final concentration of 0.25 % w/v, was applied to the plates using sterile non-
absorbent cotton wool and the plates were incubated at 37 °C for a further 2 h. Colour changes
were noted: MTT is yellow and turns purple when reduced, thereby indicating the presence of
living cells, whereas white areas on the plate indicate zones where the test bacteria have been
killed.
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4.4 RESULTS AND DISCUSSION
4.4.1 DETERMINATION OF THE ANTIBIOTIC SUSCEPTIBILITY OF
S. POLYANTIBIOTICUS SPRT
The antibiotic susceptibility of S. polyantibioticus SPRT was determined against apramycin,
hygromycin B and kanamycin in order to establish an appropriate marker to select against
untransformed cells in each transformation experiment (Table 4.4).
Table 4.4 The antibiotic susceptibility of S. polyantibioticus SPRT against various
antibiotics Antibiotic Concentration (µg/ml) Growth
Apramycin 20 -
Kanamycin 50 -
Hygromycin B 100 -
S. polyantibioticus SPRT exhibited sensitivity to apramycin, kanamycin and hygromycin B at
concentrations of 20 µg/ml, 50 µg/ml and 100 µg/ml, respectively. Apramycin was chosen as
the selectable marker for all subsequent gene disruption experiments due to the extensive
availability of E. coli-Streptomyces shuttle vectors carrying the gene for apramycin resistance.
4.4.2 THE DEVELOPMENT OF AN OPTIMISED TRANSFORMATION
PROTOCOL FOR S. POLYANTIBIOTICUS SPRT
4.4.2.1 ELECTROPRATION AND PROTOPLAST TRANSFORMATION
In order to identify the genes involved in DPO biosynthesis, an efficient DNA transformation
protocol was required for the introduction of foreign DNA into S. polyantibioticus SPRT, to be
able to disrupt genes to prove their involvement in the DPO biosynthetic pathway. Although
published transformation protocols exist for the introduction of DNA into streptomycetes, there
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
189
is no method that is generally applicable to all species. Due to the fact that S. polyantibioticus
SPRT is a novel antibiotic-producing actinomycete, the establishment of a transformation
protocol was required to make this strain amenable to genetic modifications.
Various methods for the delivery of pJN100 DNA were applied to S. polyantibioticus SPRT
cells in this study. Electroporation was the first method to be employed for transformation of
DNA into S. polyantibioticus SPRT, as it has been used in numerous Streptomyces species,
including S. coelicolor A3(2), which was used as a positive control in the present study. The
optimization of the classical electroporation methods described by Pigac & Schrempf (1995)
and Tyurin et al. (1995) proved successful for transforming plasmid DNA into S. coelicolor
A3(2). The highest number of S. coelicolor A3(2) transformants per µg of plasmid DNA was
obtained using a field strength of 7.5 kV/cm, a parallel resistor setting of 800 Ω and a time
constant of 18 ms (Figure 4.6). These optimized conditions were applied to the electroporation
of S. polyantibioticus SPRT cells without success.
Subsequently, an extensive optimization of four electroporation protocols was conducted.
Addition of various agents that affect cell wall structure to the growth medium has been shown
to enhance the electrotransformation of Gram-positive bacteria. High concentrations of glycine
(1-4 % w/v) have been used to successfully inhibit cell wall synthesis and it has been reported
that the addition of glycine and the osmotic stabilizer, sucrose, resulted in an increased
transformation efficiency of Lactococcus lactis (Papagianni et al., 2007; Holo and Nes, 1989).
Furthermore, supplementation of the growth medium with glycine and L-threonine produced
B. subtilis cells that could be electrotransformed much more efficiently at frequencies up to 2.5
x 103 transformants/µg of plasmid DNA (McDonald et al., 1995). Additionally, the
supplementation of growth media with penicillin G, which affects the cross-linking of the cell
wall in Gram-positive bacteria, was shown to enhance the transformation frequency in
Rhodococcus rhodochrous CF222 (Sunairi et al., 1996).
Initially, the filamentous growth of S. polyantibioticus SPRT proved an obstacle for all of the
methods, as the filaments aggregated. However, the addition of PEG-1000, sucrose and/or
glycine was sufficient to impair the formation of the filaments. It was therefore noted that the
most appropriate time span for electroporation was within the first 24 h of growth due to the
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
190
fact that a longer incubation time resulted in a high cell density culture containing aggregates
of intertwined hyphae, which caused arcing during electroporation.
In order to circumvent the potent restriction-modification systems present in many
Streptomyces species, all plasmid DNA used for electroporation into S. polyantibioticus SPRT
was isolated from E. coli dam- dcm- cells. Additionally, a restriction inhibitor, TypeOne
Restriction Inhibitor (Epicentre, USA), was incorporated into each electroporation reaction in
order to block the DNA binding site of type 1 R-M systems and inhibit cleavage of unmodified
DNA.
However, despite efforts to optimize an efficient electroporation protocol for
S. polyantibioticus SPRT, none proved successful. Consequently, in an effort to avoid the
problems caused by cell filaments and/or cell wall structures, protoplasts were used for the
introduction of plasmid DNA into S. polyantibioticus SPRT. Protoplast formation was achieved
after a period of 48 h in S. polyantibioticus SPRT, which was microscopically observed every
12 h up until the minimum required time for protoplast formation of 48 h, in contrast to the
rapid protoplast formation, approximately 25 min, observed in S. coelicolor A3(2). Despite
the success in preparing S. polyantibioticus SPRT protoplasts using standard procedures, they
could not be regenerated on the standard regeneration medium, R2YE (Kieser et al., 2000) or
the alternative medium, VMSO.1 (Marcone et al., 2010) under conditions where protoplasts of
other Streptomyces spp. were regenerated. Thus, it was never possible to test protoplast
transformation of S. polyantibioticus SPRT cells.
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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Figure 4.6 Effects of various electroporation parameters on the transformation efficiency of S. coelicolor
A3(2). (A) Effect of the initial voltage of the applied electrical pulse on transformation
efficiency of the electroporated cells at 25 µF and 800 Ω. (B) Effect of the external parallel
resistance on transformation efficiency of the electroporated cells at 25 µF and 7.5 kV/cm. (C)
Effect of the electrical pulse duration (time constant) on transformation efficiency of
electroporated cells at 25 µF and 7.5 kV/cm.
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14
Tran
sfo
rman
ts/µ
g D
NA
Field strength (kV/cm)
A
0
20
40
60
80
100
120
140
0 200 400 600 800 1000
Tran
sfo
rman
ts/µ
g D
NA
Parallel resistor setting
B
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20
Tran
sfo
rman
ts/µ
g D
NA
Time constant (ms)
C
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It is possible that the inability to transform DNA into S. polyantibioticus SPRT, using
electroporation, could be attributed to the inefficient expression of the heterologous apramycin-
resistance gene promoter in plasmid pJN100 when transformed into S. polyantibioticus SPRT.
The apramycin resistance gene was originally isolated from Klebsiella pneumoniae and it has
previously been reported that when the promoter being used is heterologous to the host strain,
expression may be feeble or non-existent due to unknown reasons (Wilkinson et al., 2002).
The apramycin resistance gene cassette in pJN100 was therefore replaced with the hygromycin
resistance cassette (originally isolated from Streptomyces hygroscopicus) to generate the
modified vector, pJNHYG. It was hoped that the hygromycin resistance gene would be more
efficiently expressed in S. polyantibioticus SPRT, due to the fact that it was derived from the
same genus, thereby allowing transformation and integration of pJNHYG in S. polyantibioticus
SPRT. However, no exconjugants were observed after transformation with pJNHYG,
suggesting that the problem was one of getting plasmid DNA into S. polyantibioticus SPRT
rather than one of antibiotic selection.
4.4.2.2 INTERGENERIC CONJUGATION
Intergeneric conjugation has been used to circumvent plasmid DNA transformation problems
in Streptomyces strains that are recalcitrant to protoplast transformation, most likely due to the
fact that ssDNA is transferred thereby bypassing any R-M systems (Voeikova, 1999).
Additionally, intergeneric conjugation allows for the utilization of E. coli shuttle vectors
containing the oriT function that can be used for targeted gene disruption (Kieser et al., 2000).
Two E. coli strains were used as donors in intergeneric conjugations, S17-1 and
ET12567/pUZ8002, with S. coelicolor A3(2) and S. polyantibioticus SPRT as the recipient
strains. Additionally, the conjugal ability of E. coli ET12567/pUZ8002 harbouring pOJAD2
was determined by successful mating with E. coli JM109.
Donor cultures carrying pJN100 were mated individually with S. coelicolor A3(2) and
S. polyantibioticus SPRT. The E. coli S17-1 and ET12567/pUZ8002 strains were efficient in
transferring the pJN100 plasmid to S. coelicolor A3(2) using classical conjugation methods
with conjugation frequencies of 2.1 x 10-5 and 1.83 x 10-5 exconjugants per 1 x 107 donor E.
coli cells, respectively, which is comparable to the 1 x 10-5 exconjugants obtained by Flett et
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
193
al. (1997) (Figure 4.7). However, the transfer of pJN100 into S. polyantibioticus SPRT was
only successful using E. coli ET12567/pUZ8002 as the donor strain with a conjugation
frequency of 8.3 x 10-6 exconjugants per 1 x 107 donor E. coli cells (Figure 4.7). The
unsuccessful mating between E. coli S17-1 and S. polyantibioticus SPRT may have been due
to the fact that the vigorous growth of E. coli S17-1 caused the donor strain to rapidly reach
stationary phase and thereby overgrow relative to S. polyantibioticus SPRT, before E. coli-
streptomycete mating junctions had been established (Flett et al., 1997).
In contrast to the classical conjugation methods described by Mazodier et al. (1989) and Flett
et al. (1997), whereby a glycerol spore preparation of the recipient strain was subjected to heat
treatment to induce germination and thereafter mated directly with the donor E. coli strain,
freshly germinated S. polyantibioticus SPRT spores were inoculated into liquid growth medium
for cultivation for 18 h before mating took place. Furthermore, only liquid SMC medium
containing 10.3 % sucrose produced a dispersed culture of S. polyantibioticus SPRT mycelia
that promoted the transfer of plasmid DNA across the cell wall. No exconjugants were
observed when the S. polyantibioticus SPRT cells were grown in any other medium.
In addition, it has been reported that the mycelial age of the recipient strain is a critically
important factor for the uptake of plasmid DNA in terms of competence of the recipient cells.
Luo et al. (2009) reported that Nonomuraea mycelia collected during the exponential phase
were optimal for intergeneric conjugation, whereas mycelia collected from Streptomyces
peucetius after 34 h growth were most favourable for intergeneric conjugation (Paranthaman
et al., 2003). Indeed, it was observed that S. polyantibioticus SPRT mycelia collected after 18 h
were optimal for intergeneric conjugation with E. coli ET12567/pUZ8002/pJN100 with a
conjugation frequency of 8.3 x 10-6 exconjugants per 1 x 107 donor E. coli cells (Figure 4.8).
The conjugation frequency decreased 3.3-fold to 2.5 x 10-6 after 24 h of cultivation. Mycelia
collected earlier than 18 h or later than 24 h were not suitable for conjugation as no
exconjugants were observed.
Lastly, it has been observed that the solid medium on which the intergeneric conjugation takes
place has a significant influence on the conjugation frequency in various streptomycetes (Du
et al., 2012; Guan & Pettis, 2009; Kim et al., 2008; Choi et al., 2004; Kitani et al., 2000; Flett
et al., 1997). In this study, four solid media, MS, YEME, 7H9 and ISP Medium 4, each
supplemented with either 10 mM MgCl2 or 20 mM MgCl2, were tested in order to determine
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
194
which one promoted intergeneric conjugation between E. coli ET12567/pUZ8002/pJN100 and
S. polyantibioticus SPRT the most. Exconjugants were only obtained on YEME and 7H9
media, while no exconjugants were observed on MS or ISP4 media (Figure 4.9). YEME media
supplemented with 20 mM MgCl2 proved to be optimal for the conjugation of E. coli
ET12567/pUZ8002/pJN100 and S. polyantibioticus SPRT with a conjugation frequency of 8.2
x 10-6 exconjugants per 1 x 107 donor E. coli cells, 1.1-fold higher than that of YEME
supplemented with 10 mM MgCl2 and 2.3-fold higher than 7H9 medium supplemented with
10 mM MgCl2 (conjugation frequency of 3.5 x 10-6 exconjugants per 1 x 107 donor E. coli
cells). No significant differences were observed between media supplemented with 10 mM
MgCl2 or 20 mM MgCl2.
Figure 4.7 Effect of E. coli donor on intergeneric conjugation with S. coelicolor A3(2) and
S. polyantibioticus SPRT. The data are presented as the mean ± SEM of three independent
experiments of the number of S. polyantibioticus SPRT exconjugants observed per 1 x 107 donor
E. coli cells. SC denotes S.coelicolor A3(2), SPR denotes S. polyantibioticus SPRT, S17 denotes
E. coli S17-1/pJN100 and ET denotes E. coli ET12567/pUZ8002/pJN100.
0.0E+00
5.0E-06
1.0E-05
1.5E-05
2.0E-05
2.5E-05
SC + S17 SC + ET SPR + S17 SPR + ET
Co
nju
gati
on
fre
qu
ency
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Figure 4.8 Effect of the mycelial age of the recipient strain, S. polyantibioticus SPRT, on intergeneric
conjugation with E. coli ET12567/pUZ8002/pJN100. The data are presented as the mean ± SEM of three independent experiments of the number of S. polyantibioticus SPRT exconjugants
observed per 1 x 107 donor E. coli cells.
0.0E+00
1.0E-06
2.0E-06
3.0E-06
4.0E-06
5.0E-06
6.0E-06
7.0E-06
8.0E-06
9.0E-06
5 12 18 24 48 72
Co
nju
gati
on
fre
qu
ency
Mycelial age (Hours)
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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Figure 4.9 Effect of mycelial solid growth medium on intergeneric conjugation between E. coli
ET12567/pUZ8002/pJN100 and S. polyantibioticus SPRT. The data are presented as the mean
± SEM of three independent experiments of the number of S. polyantibioticus SPRT
exconjugants observed per 1 x 107 donor E. coli cells.
4.4.3 GENE DISRUPTION USING OPTIMIZED INTERGENERIC
CONJUGATION METHOD
In order to determine which NRPS A domain is involved in DPO biosynthesis, in addition to
testing the involvement of the genes encoding the phenylacetate-CoA ligase, thioesterase,
cinnamate-CoA ligase, D-lactate dehydrogenase and acyl-CoA synthetase (as well as the
putative NRPS Cy domain) in DPO synthesis, it was decided that all of these genes could be
knocked-out in S. polyantibioticus SPRT using the method of homologous recombination.
The shuttle vector, pOJ260, was genetically manipulated for use in the gene disruption
experiments. Briefly, fragments of the genes encoding the A domain, Cy domain,
phenylacetate-CoA ligase, thioesterase, cinnamate-CoA ligase, D-lactate dehydrogenase and
0.0E+00
1.0E-06
2.0E-06
3.0E-06
4.0E-06
5.0E-06
6.0E-06
7.0E-06
8.0E-06
9.0E-06
YEME + 20mM MgCl2
YEME + 10mM MgCl2
MS + 10mMMgCl2
MS + 20mMMgCl2
7H9 + 10mM MgCl2
7H9 + 20mM MgCl2
ISP4 + 10mM MgCl2
ISP4 + 20mM MgCl2
Co
nju
gati
on
fre
qu
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Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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acyl-CoA synthetase were individually cloned into pOJ260 and transformed into E. coli
ET12567/pUZ8002 for use in subsequent intergeneric conjugation with S. polyantibioticus
SPRT using the optimized, strain-specific conjugation method described in section 4.4.2.2.
The gene disruption experiments were based on the premise that the plasmid, pOJ260, carrying
a homologous region to a gene in S. polyantibioticus SPRT, would integrate into the genome
by a single crossover event and thereby disrupt the target gene (Figure 4.10). It would then be
possible to infer the gene’s involvement in the production of DPO, should the mutant strain
lose the ability to produce DPO. In order to detect the production of DPO after each respective
gene knockout, DPO was extracted from the resulting mutants and subjected to bioautography
against M. aurum A+. A loss in the detection of activity of the extract against M. aurum A+
indicated a loss of production of DPO and thereby confirmed the gene’s involvement in DPO
biosynthesis.
Fragments of the A domains designated AD2, A18, A16, A28, A7 and A99, in addition to the
genes encoding the phenylacetate-CoA ligase (PAAK), the Cy domain (CYC), acyl-CoA
synthetase (ACY), D-lactate dehydrogenase (LAC), cinnamate-CoA ligase (CIN) and
thioesterase (THI), were PCR-amplified from the S. polyantibioticus SPRT genome and
individually cloned into pOJ260. The resulting plasmids were designated pOJAD2, pOJA18,
pOJA16, pOJA28, pOJA7, pOJPAAK, pOJCYC, pOJA99, pOJACY, pOJLAC, pOJCIN and
pOJTHI, and were individually transformed into E. coli ET12567/pUZ8002 and introduced
into S. polyantibioticus SPRT via intergeneric conjugation to yield the mutant strains S.
polyantibioticus ∆AD2, S. polyantibioticus ∆A18, S. polyantibioticus ∆A16, S.
polyantibioticus ∆A28, S. polyantibioticus ∆A7, S. polyantibioticus ∆PAAK, S.
polyantibioticus ∆CYC, S. polyantibioticus ∆A99, S. polyantibioticus ∆ACY, S.
polyantibioticus ∆LAC, S. polyantibioticus ∆CIN and S. polyantibioticus ∆THI, respectively.
The location of DNA fragments used to make knock-out constructs within the genes they target,
according to Figure 4.10, is provided in Appendix C.
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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Figure 4.10 Schematic diagram representing the strategy employed in the construction of a theoretical gene
A disruption mutant (A). Gene disruption was achieved by inactivation of the target gene via
single crossover homologous recombination. The horizontal black arrows represent the position
of the binding sites of the PCR primers that were designed to confirm the integration of the
plasmid DNA, carrying the target gene of interest, into the wild-type chromosome. GSP refers
to the gene specific primer, depending on which target gene had been integrated into the S.
polyantibioticus SPRT genome. The GSP primer was used in conjunction with either the
POJFWD or POJREV primer. The binding positions of the POJFWD and POJREV primers on
the recombinant pOJ260 plasmid before integration of the whole plasmid into the genome is
also depicted. The apramycin resistance gene is represented by the abbreviation, apr. The
nucleotide sequences of the regions represented by the vertical arrows is provided in Appendix
C for each knock-out construct. (B). The red line indicates the target gene that has been cloned
into the multiple cloning region of pOJ260 and the numbers indicate the exact binding positions
on the plasmid according to the pOJ260 plasmid map (Figure 4.3).
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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Confirmation of the integration of the vector carrying the target gene of interest into the genome
of each mutant strain was determined by PCR amplification across the junctions of each copy
of the mutant gene derived from homologous recombination using the POJFWD primer and
the respective reverse gene specific primer (Figure 4.11-4.12). Amplification using the forward
gene specific primer of interest and POJREV in combination also confirmed the integration of
the vector carrying the target gene of interest (data not shown). Due to the binding positions of
the POJFWD and POJREV primers, the amplified products were 72 bp longer than the
amplified product obtained using the gene specific primer set, when using the POJFWD primer
in combination with the respective reverse target gene specific primer and 97 bp longer using
the POJREV primer in combination with the forward target gene specific primer (Table 4.5).
Additionally, the full open reading frame of the acyl-CoA synthetase was PCR-amplified and
cloned into the plasmid vector pJN100 to generate pJNACY, which was transformed into E.
coli ET12567/pUZ8002 and introduced into S. polyantibioticus ∆ACY via intergeneric
conjugation in order to complement the acyl-CoA synthetase mutant strain, S. polyantibioticus
∆ACY.
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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Figure 4.11 Gel electrophoresis of PCR amplified A domains from S. polyantibioticus SPRT using the
POJFWD and A7R primer set. Lanes: 1- λ-PstI molecular marker, 2- A18 amplification product
from S. polyantibioticus ∆18 gDNA, 3- A28 amplification product from S. polyantibioticus ∆28
gDNA, 4- A16 amplification product from S. polyantibioticus ∆16 gDNA, 5- A99 amplification
product from S. polyantibioticus ∆99 gDNA, 6- A7 amplification product from S.
polyantibioticus ∆7 gDNA, 7- AD2 amplification product from S. polyantibioticus ∆AD2
gDNA.
11.54
5.08
2.84
1.70
0.81
0.55
kb
1.16
1 2 3 4 5 6 7
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Figure 4.12 Gel electrophoresis of PCR amplified target genes from S. polyantibioticus SPRT using the
POJFWD and reverse gene specific primers in combination. Lanes: 1- λ-PstI molecular marker,
2- CIN amplification product from S. polyantibioticus ∆CIN gDNA, 3- LAC amplifcation
product from S. polyantibioticus ∆LAC gDNA, 4- CYC amplification product from S.
polyantibioticus ∆CYC gDNA, 5- PAAK amplification product from S. polyantibioticus
∆PAAK gDNA, 6- THI amplification product, from S. polyantibioticus ∆THI gDNA.
11.54
5.08
2.84
1.70
0.81
0.55
kb
1.16
1 2 3 4 5 6
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Table 4.5 PCR amplification product sizes of target genes using POJFWD and POJREV
primers Target gene Product size using POJFWD
and gene specific reverse primer
(bp)
Product size using gene specific
forward primer and POJREV
(bp)
A18 742 767
A16 771 796
A28 780 805
AD2 794 817
A7 802 827
A99 792 817
CIN 721 746
LAC 680 705
THI 545 570
CYC 1052 1149
PAAK 588 613
ACY 993 1017
4.4.4 ISOLATION OF DPO FROM S. POLYANTIBIOTICUS SPRT AND
CONFIRMATION OF ITS ACTIVITY AGAINST M. AURUM A+
The antibacterial compound, DPO, was isolated from the fermentation broth and mycelial mass
of S. polyantibioticus SPRT. The activity of the compound was detected through
bioautography, displaying an Rf of 0.88 with M. aurum A+ as the test organism.
Chemically-synthesized 2,5-diphenyloxazole was used as a positive control for the detection
of the bioactivity and its Rf value correlated with the biologically-produced compound (Figure
4.13). Furthermore, the solvent used in the purification of DPO, toluene, was employed as a
negative control in the detection of bioactivity in order to ensure that no contaminating
compounds with similar bioactivity to DPO were present.
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Reverse-phase HPLC was employed to corroborate the results obtained from the bioautography
and the retention times measured at 280 nm of the biologically-produced DPO and chemically-
synthesized DPO were identical. The results of the TLC-bioautography analysis and reverse-
phase HPLC confirmed the production of DPO by S. polyantibioticus SPRT and its activity
against M. aurum A+.
Figure 4.13 TLC bioautography and RP-HPLC analysis of the biologically-synthesized DPO sample
isolated from S. polyantibioticus SPRT and the chemically synthesized DPO sample. (A) TLC
plates stained with MTT showing that both 1) the chemically synthesized DPO sample and 2)
the biologically-synthesized DPO sample had antibacterial activity and an Rf of 0.88 in the
solvent system used (100% ethyl acetate). (B) RP-HPLC analysis depicting the retention time,
29.65 min, of the chemically synthesized DPO sample measured at 280 nm. (C) RP-HPLC
analysis depicting the retention time, 29.65 min, of the biologically-synthesized DPO sample
measured at 280 nm.
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4.4.5 TLC BIOAUTOGRAPHY ANALYSIS TO DETERMINE THE PUTATIVE
INVOLVEMENT OF THE TARGET GENES IN DPO BIOSYNTHESIS
In order to directly determine whether the A domains designated AD2, A18, A16, A28, A7 and
A99, as well as the genes encoding the phenylacetate-CoA ligase (PAAK), the Cy domain
(CYC), acyl-CoA synthetase (ACY), D-lactate dehydrogenase (LAC), cinnamate-CoA ligase
(CIN) and thioesterase (THI), are involved in the biosynthesis of DPO, each gene was disrupted
and the resulting mutant strain, lacking a functional copy of the target gene, was assayed for
activity against M. aurum A+. A loss in activity would suggest the gene’s involvement in DPO
biosynthesis.
The DPO purification method was carried out on cultures of strains S. polyantibioticus SPRT,
S. polyantibioticus ∆AD2, S. polyantibioticus ∆A18, S. polyantibioticus ∆A16, S.
polyantibioticus ∆A28, S. polyantibioticus ∆A7, S. polyantibioticus ∆PAAK, S.
polyantibioticus ∆CYC, S. polyantibioticus ∆A99, S. polyantibioticus ∆ACY, S.
polyantibioticus ∆LAC, S. polyantibioticus ∆CIN and S. polyantibioticus ∆THI. Each sample
was subjected to UV spectrophotometry which confirmed the presence of a single peak at a
wavelength of 280 nm in the samples from S. polyantibioticus SPRT, S. polyantibioticus ∆AD2,
S. polyantibioticus ∆A18, S. polyantibioticus ∆A16, S. polyantibioticus ∆A28, S.
polyantibioticus ∆A7, S. polyantibioticus ∆PAAK, S. polyantibioticus ∆LAC, S.
polyantibioticus ∆CIN and S. polyantibioticus ∆THI. In contrast, the samples from strains S.
polyantibioticus ∆CYC, S. polyantibioticus ∆A99 and S. polyantibioticus ∆ACY displayed a
reading of zero at a wavelength of 280 nm, thereby indicating that these strains had lost the
ability to produce DPO.
TLC-bioautography analysis was performed on the extracts from each strain, which showed
that S. polyantibioticus ∆AD2, S. polyantibioticus ∆A18, S. polyantibioticus ∆A16, S.
polyantibioticus ∆A28, S. polyantibioticus ∆A7, S. polyantibioticus ∆PAAK, S.
polyantibioticus ∆LAC, S. polyantibioticus ∆CIN and S. polyantibioticus ∆THI all had activity
against M. aurum A+ (Figures 4.14 and 4.15). This showed that disruption of these genes did
not abolish DPO production, which indicated that none of these genes is involved in DPO
biosynthesis. However, the extracts from strains S. polyantibioticus ∆CYC (Figure 4.16), S.
polyantibioticus ∆A99 (Figure 4.17) and S. polyantibioticus ∆ACY (Figure 4.18) showed no
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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activity against M. aurum A+, implying that the disruption of these genes resulted in the strains
losing the ability to produce DPO.
Figure 4.14 TLC plates stained with MTT showing the bioautographic analysis of DPO isolated from lane
B) S. polyantibioticus ∆AD2, lane C) S. polyantibioticus ∆A18, lane D) S. polyantibioticus
∆A16, lane E) S. polyantibioticus ∆A28 and lane F) S. polyantibioticus ∆A7, where lane A) is
chemically-synthesized DPO acting as a positive control.
Additionally, the introduction of pJNACY into S. polyantibioticus ∆ACY via intergeneric
conjugation in a complementation experiment, resulted in the restoration of DPO biosynthesis
and the ability to inhibit M. aurum A+ (Figure 4.18).
These results demonstrated the involvement of the Cy domain encoded by gene SPR_53040,
the A domain encoded by gene SPR_53060 and the acyl-CoA synthetase encoded by gene
SPR_52860 in the production of DPO. Furthermore, these results excluded the involvement
of the cinnamate-CoA ligase encoded by gene SPR_60150, the putative D-lactate
dehydrogenase encoded by gene SPR_60250 and the TE domain encoded by gene SPR_53090
A B C D E F
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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in the biosynthesis of benzoic acid, which is proposed to be required for DPO biosynthesis. It
also showed that none of the other A domains investigated is involved in DPO biosynthesis.
Figure 4.15 TLC plates stained with MTT showing the bioautographic analysis of DPO isolated from lane
B) S. polyantibioticus ∆PAAK, lane C) S. polyantibioticus ∆LAC, lane E) S. polyantibioticus
∆CIN and lane F) S. polyantibioticus ∆THI, where lanes A) and D) are chemically-synthesized
DPO acting as a positive control.
A B C D E F
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Figure 4.16 TLC plates stained with MTT showing the bioautographic analysis of DPO isolated from lane
B) S. polyantibioticus SPRT, lane C) S. polyantibioticus ∆CYC, where lane A) is chemically-
synthesized DPO acting as a positive control.
A B C
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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Figure 4.17 TLC plates stained with MTT showing the bioautographic analysis of DPO isolated from lane
B) S. polyantibioticus SPRT, lane C) S. polyantibioticus ∆A99, where lane A) is chemically-
synthesized DPO acting as a positive control.
A B C
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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Figure 4.18 TLC plates stained with MTT showing the bioautographic analysis of DPO isolated from lane
B) S. polyantibioticus ∆ACY, lane C) S. polyantibioticus SPRT and lane D) S. polyantibioticus
PJNACY, where lane A) is chemically-synthesized DPO acting as a positive control.
A B C D
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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4.5 CONCLUSION An effective intergeneric conjugation method for the introduction of plasmid DNA from E. coli
ET12567 into S. polyantibioticus SPRT was developed and optimized. This method of
transformation allowed for the identification and targeted manipulation of the biosynthetic
pathway involved in DPO production. S. polyantibioticus mutant strains lacking a functional
copy of the putative Cy domain encoded by gene SPR_53040, the A domain encoded by gene
SPR_53060 and the acyl-CoA synthetase encoded by gene SPR_52860 exhibited a loss in the
production of DPO and were thereby identified as putative members of the DPO biosynthetic
pathway.
Due to the fact a gene encoding an acyl-CoA synthetase was not part of the initial hypothesis
on how DPO is produced, it was deemed necessary to amend the original theory. The
introduction of the revised hypothesis on how DPO is produced by S. polyantibioticus SPRT,
including an hypothesis on how benzoic acid is synthesized, is described in the following
chapter.
Chapter 4 – Development of a transformation protocol for S. polyantibioticus SPRT and gene disruption experiments
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218
CHAPTER 5
GENERAL DISCUSSION
5.1 Introduction ................................................................................................................ 219
5.2 Identification and characterisation of the genes involved in joining benzoic acid and
phenylalanine/3-hydroxyphenylalanine in S. polyantibioticus SPRT to create a 2,5-
disubstituted oxazole ................................................................................................. 221
5.3 Identification of the genes responsible for the biosynthesis of benzoic acid in
S. polyantibioticus SPRT ........................................................................................... 228
5.4 Proposed pathway for the biosynthesis of DPO in S. polyantibioticus SPRT ........... 237
5.5 Conclusion and future work ....................................................................................... 240
5.6 Reference list ............................................................................................................. 241
Chapter 5 – General Discussion
219
CHAPTER 5
GENERAL DISCUSSION
5.1 Introduction
Natural products, such as bacterial secondary metabolites, have proven to be valuable sources
of novel compounds exhibiting a diverse array of biological activities. Indeed, due to their
diverse and complex chemical structures, bacterial secondary metabolites have demonstrated
their use as pharmacologically active compounds that continue to serve medicine well by
offering novel leads for novel therapeutic agents (Harvey, 1999; Bernan et al., 1997).
Additionally, the great structural and chemical diversity of natural products makes them well-
suited for manipulation by chemical and/or genetic means to generate semi-synthetic drugs
with improved antimicrobial and pharmacological properties (Ashforth et al., 2010). The first-
line anti-TB drug, rifampicin, for example, was developed from the rifamycins, which are
produced by members of the actinobacterial genus, Amycolatopsis (Wright, 2012).
The need for novel antibiotics with novel mechanisms of action has become urgent with the
increased occurrence of bacterial resistance to known antibiotics and the emergence of new
and old diseases (especially in the case of tuberculosis) (Bérdy, 2005).
It has been estimated that only 1–3 % of all antibiotics have been discovered, meaning that
there is a high likelihood of identifying new antibiotic molecules from bacteria (and particularly
actinobacteria) through a taxonomy-guided exploration of bacterial biodiversity. Certainly, the
bacteria belonging to the suborders Streptomycineae (e.g. Streptomyces), Micromonosporineae
(e.g. Micromonospora), Pseudonocardineae (e.g. Amycolatopsis) and Streptosporangineae
(e.g. Planobispora) (all in the order Actinomycetales; class Actinobacteria) are the most
prolific producers of novel antimicrobial antibiotics (Ashforth et al., 2010). To gain access to
the biodiversity in these (and other) actinobacterial suborders, it is important to isolate
Chapter 5 – General Discussion
220
actinobacteria from many different environments, e.g. terrestrial soil and marine sediments, as
well as from plants and animals.
Actinobacterial genomes (particularly those of the actinomycetes, such as Streptomyces) are
larger than those of most other bacteria, possessing a high capacity for the synthesis of
secondary metabolites and employing a substantial fraction of coding capacity (5–10 %) for
the production of mostly cryptic secondary metabolites (Baltz, 2008). The sequenced
actinobacterial genomes are a valuable resource, which have revealed that actinobacteria have
the biosynthetic potential to make far more natural products than was realised (Ashforth et al.,
2010). The taxonomic diversity covered by these sequenced genomes has revealed that the
more bacterial genomes that are sequenced, the more new gene families are discovered (Wu et
al., 2009). Thus, identifying taxonomic diversity reveals new genetic diversity and therefore
new biosynthetic capabilities.
In light of the taxonomy-guided bacterial bioprospecting approach described above,
S. polyantibioticus SPRT was isolated as part of an antibiotic-screening programme and
identified as the producer of several antibiotics (Le Roes-Hill & Meyers, 2009; Le Roes, 2005).
One of these antibiotics possessed antitubercular activity and was identified as DPO (Le Roes,
2005). In this study, the S. polyantibioticus SPRT genome was sequenced and explored in order
to identify the biosynthetic pathway involved in DPO biosynthesis and thereby take the first
step towards the generation of derivatives of DPO by combinatorial biosynthesis.
Combinatorial biosynthesis is a powerful way to generate antibiotic derivatives with greater
antibacterial activity and/or improved pharmacokinetic properties. It has advantages over the
chemical derivatisation of antibiotic molecules, since the bacterial biosynthetic enzymes carry
out site-specific and enantiomer-specific reactions, by-passing the need to protect reactive
functional groups in each reaction step in the chemical approach. This ensures that only the
desired product is produced, which enhances the product yield.
In order to determine whether the proposed biosynthetic pathway is correct, the genes coding
for benzoic acid synthesis and the DPO NRPS had to be identified in the S. polyantibioticus
SPRT genome. The research aims of this study, as described in Chapter 1, are discussed in the
following sections within the context of the original hypothesis for DPO biosynthesis in S.
polyantibioticus SPRT.
Chapter 5 – General Discussion
221
5.2 Identification and characterisation of the genes involved in joining
benzoic acid and phenylalanine/3-hydroxyphenylalanine in S.
polyantibioticus SPRT to create a 2,5-disubstituted oxazole
Based on the structure of DPO, a biosynthetic scheme for the synthesis of this molecule, in
which an NRPS condenses a molecule of benzoic acid with 3-hydroxyphenylalanine (also
known as 3-phenylserine) to form an amide bond between them, was proposed (Chapter 1).
This intermediate, known as N-benzoyl-β-hydroxyphenylalanine (Figure 5.1), is subsequently
converted to a diphenyloxazole derivative by heterocyclization across the amide bond, after
which a final decarboxylation step leads to DPO. An NRPS is believed to be the core enzyme
in the synthesis of DPO due to the presence of an oxazole in the DPO structure.
Figure 5.1 The proposed amide-bond-containing precursor to DPO, N-benzoyl-β-hydroxyphenylalanine.
The atoms joined by the three red bonds (i.e. running from the carbonyl C atom of benzoic acid
to the hydroxyl C atom of 3-hydroxyphenylalanine) are involved in forming a 5-membered
heterocyclic ring, i.e. an oxazole.
Initial efforts to isolate and identify the DPO biosynthetic gene cluster in S. polyantibioticus
SPRT identified twelve unique NRPS A domains. The specificities of these A domains were
identified using NRPSpredictor2 (Rausch et al., 2005). This approach has been used in
numerous studies and is considered accurate. However, there have been examples of substrate-
Chapter 5 – General Discussion
222
specificity codes for which the predicted substrate specificity does not correspond to the
activated amino acid identified in vivo (Lombó et al., 2006). A study by McQuade et al. (2009)
suggested that, in addition to the 10 key residues of the binding pocket, there may be other
residues involved in controlling the substrate amino acid activated by an A domain.
In this study, the A domain contained within the inserts of the identical clones pGEMA-18 and
pGEMA-66 was predicted to be specific for the activation of phenylalanine, while two A
domains contained within the inserts of the clones pGEMA-5/pGEMA-7/pGEMA-
12/pGEMA-22 and pGEMA-36/pGEMA-51/pGEMA-63, where predicted to be specific for
serine. Serine is a common starter unit for the biosynthesis of oxazoles. However, if serine
were used in DPO biosynthesis, it would require the addition of a phenyl group to the oxazole
structure (Roy et al., 1999). Importantly, β-phenylation of an activated serine has not yet been
reported to occur in bacteria and it therefore seems more likely that β-hydroxylation of an
activated phenylalanine residue by a P450 monooxygenase would be the mechanism involved
in DPO biosynthesis. Nevertheless, it is possible that a β-phenylation reaction is used by S.
polyantibioticus SPRT (Le Roes, 2005).
Annotation of the S. polyantibioticus SPRT draft genome sequence revealed that the A domain
insert in clone pGEMA-18/pGEMA-66 is encoded by gene SPR_48330. This gene was
predicted by antiSMASH 3.0 to be involved in the biosynthesis of an antimycin-type antibiotic
and was therefore dismissed as being a member of the DPO biosynthetic cluster. Similarly,
the A domains predicted to activate serine and contained within the clones pGEMA-5/pGEMA-
7/pGEMA-12/pGEMA-22 and pGEMA-36/pGEMA-51/pGEMA-63, are encoded by the genes
SPR_56140 and SPR_56150, respectively. Due to their predicted involvement in the
biosynthesis of a complex PKS/NRPS hybrid secondary metabolite, genes SPR_56140 and
SPR_56150 were deemed unlikely to be involved in DPO biosynthesis. Nevertheless, the A
domains designated A-7 and A-18 (Chapter 2), in addition to the AD-2, A-16 and A-28
domains encoded by the genes SPR_6340, SPR_28920 and SPR_6040, respectively, were
disrupted by homologous recombination in S. polyantibioticus SPRT. Subsequent
bioautography experiments confirmed that genes SPR_6340, SPR_28920 and SPR_6040 are
not involved in DPO biosynthesis, as inactivation of these genes did not abolish DPO
production by the mutant strains.
Chapter 5 – General Discussion
223
Conversely, the A domain, A-99, encoded by SPR_53060 was confirmed as a putative member
of the S. polyantibioticus SPRT DPO biosynthetic pathway due to the fact that an
S. polyantibioticus mutant strain lacking a functional copy of this A domain exhibited a loss of
production of DPO. The specificity prediction of this A domain was however inconclusive, as
the software tools predicted specificity for both phenylalanine and tryptophan (Chapter 3).
Nevertheless, the relaxed substrate specificity of the A domains in xenematide A biosynthesis
has demonstrated the ability of A domains for aromatic amino acids to accept either tryptophan
or phenylalanine as substrates. Furthermore, Schaffer & Otten (2009) demonstrated in the
tyrocidine A biosynthetic pathway that although A domains are selective for specific substrates,
many of these domains possess the ability to adenylate a number of different substrates. Thus,
the A domain encoded by SPR_53060 might accept phenylalanine or 3-hydroxyphenylalanine
as a substrate in the biosynthesis of DPO.
However, if 3-hydroxyphenylalanine is not the starter molecule in DPO biosynthesis, it is
possible that an alternative starter molecule such as 1-phenyl-2-aminoethanol could be used in
DPO biosynthesis (Figure 5.2). Indeed, 2-aminoethanol (ethanolamine) is a compound derived
from cell membranes that certain bacteria can utilize as a source of carbon and/or nitrogen
(Garsin, 2010). It is also possible that S. polyantibioticus SPRT could utilize glycine to
synthesize 2-aminoethanol by reduction of the carboxyl group. However, a phenylation
reaction would need to occur to generate 1-phenyl-2-aminoethanol.
It is evident that, due to the weak predictions of the software tools and the contradictory results,
the substrate specificity of the A domain encoded by SPR_53060 can only be elucidated by
experimental analysis. Due to the fact that A domains consume ATP and release PPi during
the activation of their substrates, and because the activation reaction is reversible, A domain
selectivity can be assayed using the conventional ATP-32PPi isotope exchange assay in vitro.
During this assay, the A domain of interest is incubated with the potential substrate, ATP, Mg2+
and 32P-labeled PPi. During the reverse activation reaction, 32P from the labeled PPi is
incorporated into ATP if the amino acid(s) present in the assay is/are activated by the A domain.
The 32P-ATP can then be separated via adsorption to activated charcoal. The amount of
generated 32P-ATP is proportional to the substrate activation reaction (Phelan et al., 2009;
Linne & Marahiel, 2004). However, Phelan et al. (2009) developed a non-radioactive mass
spectrometry (MS)-based PPi exchange assay that is able to measure the consumption of
Chapter 5 – General Discussion
224
γ-18O4-labelled ATP and the formation of 16O4-ATP by the isotopic back exchange of excess
unlabelled PPi. The resulting mass shifts are detected by MALDI-TOF-MS and the activity of
the A domain can therefore be measured. Incubation with different substrates and a comparison
of each substrate’s ATP-PPi exchange rate allows for the determination of the substrate
specificity of the A domain under scrutiny. In contrast to the issues observed in the
conventional systems, this exchange assay provides a rapid, sensitive and reproducible means
to measure A domain specificity (Phelan et al., 2009). This technique has been used
successfully to determine the A domain specificity in studies of Streptomyces refuinius (Phelan
et al., 2009) and Streptomyces coeruleorubidus NRRL 18370 (Zhang et al., 2010), thereby
providing an alternative option for the substrate prediction of A domains in future studies of S.
polyantibioticus SPRT.
Additionally, the A domain of the acyl-CoA synthetase encoded by SPR_52860 was predicted
to be specific for the activation of an aromatic substrate such as 2,3-dihydroxybenzoic acid (or
a derivative thereof), phenylalanine or tryptophan (Chapter 3). This gene was identified as a
member of the DPO biosynthetic pathway via the gene disruption and complementation
experiments described in Chapter 4.
The family of coenzyme A ligases includes the A domains of NRPSs, firefly luciferase and
acyl- and aryl-CoA synthetases, such as the acyl-CoA synthetase encoded by SPR_52860.
Reactions catalysed by this family of enzymes proceed in two steps in which the substrate
reacts with ATP to form an acyl-adenylate intermediate with the simultaneous release of
pyrophosphate. Thereafter, the adenylate group is replaced by CoA, accompanied by the
release of AMP (Gulick, 2009). The CoA-ligases are known to initiate β-oxidation via the
activation of fatty acids and are important in the biosynthesis of natural products such as the
penicillins (Koetsier et al., 2009) and lignin (Anterola & Lewis, 2002) (Figure 5.3).
Chapter 5 – General Discussion
225
Figure 5.2 Proposed alternative route to DPO biosynthesis in S. polyantibioticus SPRT. A molecule of
benzoic acid (A) is condensed with 1-phenyl-2-aminoethanol (B) to form an ester bond between
them. Subsequent heterocyclization and decarboxylation of the intermediate, O-benzoyl-1-
phenyl-2-aminoethanol (C), would lead to DPO (D).
As the acyl-CoA synthetase encoded by SPR_52860 was identified as a member of the DPO
biosynthetic pathway, it would activate its substrate, benzoic acid, as a CoA-thioester.
Thereafter, the first reaction in DPO biosynthesis would proceed via amide bond formation
between an aminoacyl-bound PCP-thioester and the CoA-thioester. Although the activation of
a Cy domain substrate as a CoA-thioester instead of a PCP-Ppant-thioester is unusual, it has
been reported in the biosynthesis of congocidine (Juguet et al., 2009), the calcium-dependent
antibiotics (Hojati et al., 2002) and fengycin (Steller et al., 1999). It is interesting to note that
the aspartate residue (D235 according to the GrsA-PheA numbering convention) that is
Chapter 5 – General Discussion
226
conserved in the majority of A domains and which is involved in the binding of the α-amino
acid substrate is replaced by a glycine in the substrate specificity binding pocket of the A
domain encoded by SPR_52860. It is therefore suggested that the substrate of the SPR_52860
A domain does not possess an α-amino group, which would support the hypothesis that an
aromatic acid, such as benzoic acid, is indeed activated.
Figure 5.3 Examples of reactions catalysed by members of the family of adenylate-forming enzymes. (A)
The activation of the pencillin G side chain phenylacetic acid is catalysed by a phenylacetate-
CoA ligase in Penicillium chrosygenum (adapted from Koetsier et al., 2011). (B) The activation
of benzoic acid for DPO biosynthesis is believed to be catalysed by an acyl CoA-synthetase in
S. polyantibioticus SPRT.
Although, in the initial experiments described in Chapter 2, a Cy domain could not be amplified
from the S. polyantibioticus SPRT genome, a phylogenetic analysis of all of the identified C
domains within the S. polyantibioticus SPRT draft genome provided insight into the degree of
evolutionary relatedness of these domains to well-characterised reference Cy domains. The
domains encoded by SPR_53060, SPR_53040, SPR_6230, SPR_6240 and SPR_6360 shared
the highest degree of evolutionary relatedness to the reference Cy domains and an analysis of
the conserved signature motifs within these domains identified SPR_53040 and SPR_53060 as
displaying an unusual putative tandem condensation/heterocyclization function in DPO
biosynthesis. This was deduced because SPR_53040 lacks the catalytic second aspartate
Chapter 5 – General Discussion
227
residue found in the Cy domain consensus motif, whereas SPR_53060 lacks the first Cy domain
aspartate residue.
It has been demonstrated that the second aspartate residue in the Cy domain motif D-x-x-x-x-
D-x-x-S motif is critical for catalytic Cy activity, however the role of the first aspartate is still
unclear. Mutation of the first aspartate residue to alanine resulted in the elimination of the
activity in the first Cy domain of the yersiniabactin synthetase subunit HMWP2, while a
comparable mutation in the Cy domain of the vibriobactin NRPS module VibF resulted in only
a 10-fold reduction in activity (Marshall et al., 2002; Keating et al., 2000). The significance
of the first aspartate residue of the consensus motif was also examined via an alanine mutation
in the NRPS subunit EntF of enterobactin synthetase, where activity was only diminished 2-
fold in comparison to the wild type strain. The authors surmised that this result implicates the
residue as critical but not essential for heterocycle formation, with its importance varying
according to the synthetase and substrate in question (Kelly et al., 2005).
The serine residue found in the D-x-x-x-x-D-x-x-S motif is not strictly conserved among Cy
domains and a mutation of the serine residue to alanine in the yersiniabactin synthetase subunit
HMWP2 Cy domain resulted in no effect on heterocycle formation. This suggests that the lack
of a serine residue in the signature motif found in the putative Cy domain encoded by
SPR_53040 is of limited significance (Keating et al., 2000).
Peptide bond formation and heterocyclization usually depend on the action of a single Cy
domain. However, a situation where these two reactions are split between two Cy domains
was observed in the biosynthesis of the oxazoline-containing siderophore vibriobactin in Vibrio
cholerae. Such a split-function Cy-domain pair is believed to be involved in the DPO
biosynthetic pathway. The NRPS responsible for the formation of vibriobactin consists of an
unusual Cy-Cy tandem arrangement, where the first domain is responsible for heterocyclization
and the second domain is responsible for condensation (Schwarzer et al., 2003; Marshall et al.,
2002).
Additionally, in the biosynthesis of holomycin by Streptomyces clavuligerus, a tridomain
module with the organization Cy-A-PCP, together with a lone C domain, are proposed to be
responsible for the selection, activation, heterocyclization and oxidation of two cysteine
Chapter 5 – General Discussion
228
residues in the biosynthesis of the final product. This further demonstrates the flexibility of
NRPS systems, where instead of a Cy domain simply replacing the C domain as observed in
conventional NRPS elongation modules for oxazoles and thiazoles, a pair of domains functions
in tandem (Li & Walsh, 2010).
5.3 Identification of the genes responsible for the biosynthesis of benzoic
acid in S. polyantibioticus SPRT
In bacteria, only a handful of benzoic acid derived metabolites have been described, seemingly
due to the rarity of PAL in these organisms. Indeed, despite the plant-like biosynthesis of
benzoyl-CoA described in “S. maritmus” strain DSM 41777T, where this rare starter unit is
involved in the production of the structurally novel polyketide enterocin (Hertweck & Moore,
2000), unsubstituted benzoyl units have only been found in benzoyl α-L-rhamnopyranoside
produced by Streptomyces griseoviridis Tü 3634 (Hoffman & Grond, 2004), as well as in the
structures of aestivophoenin A (Kunigami et al., 1998) and wailupemycin (Hertweck & Moore,
2000), all isolated from Streptomyces spp., and in the myxobacterial molecules soraphen (Hill
et al., 2003), thiangazole and crocacin (Jansen et al., 1992). In stark contrast to this, the
occurrence of benzoyl units is common in secondary metabolites produced by plants and fungi,
such as in the cancer drug taxol (Rohr, 1997) and the fungicide strobilurin (Nerud et al., 1982).
In addition to the aerobic benzoyl-CoA biosynthetic process catalysed by PAL in “S. maritmus”
strain DSM 41777T, there is evidence for two PAL-independent pathways resulting in the
biosynthesis of benzoic acid, namely, via: 1) the anaerobic degradation of L-phenylalanine
described in the denitrifying bacterium Thauera aromatica (Breese et al., 1998; Schneider et
al., 1997) and 2) directly from shikimate (Hoffman & Grond, 2004). Additionally, soraphen
A biosynthesis in S. cellulosum is an example of an aerobic non-PAL pathway, which begins
with a phenyl side group that is derived from phenylalanine, but the exact pathway for the
generation of benzoic acid is unknown. It has been proposed that carboxybenzoyl-CoA is an
intermediate in the biosynthesis of benzoic acid in S. cellulosum (Schupp et al., 1995, Ligon et
al., 2002).
Chapter 5 – General Discussion
229
In this study, a number of approaches to obtaining the genes responsible for the biosynthesis
of benzoic acid in S. polyantibioticus SPRT were explored. The first approach focused on the
search for an orthologue of the encP gene, which encodes PAL. Despite utilizing
‘S. maritimus’ DSM 41777T as a positive control, no encP orthologue was detected in the
S. polyantibioticus SPRT genome by PCR amplification and Southern blotting. However, a
HAL, which is highly homologous to PAL and catalyzes the analogous deamination of
histidine to trans-urocanate in prokaryotes, thereby initiating histidine degradation (Michal,
1999), was detected in the S. polyantibioticus SPRT genome using PCR amplification and
sequencing. Both HAL and PAL enzymes are also homologous to tyrosine ammonia lyase
(TAL), an enzyme commonly found in plants (Kyndt et al., 2002). All three enzymes contain
the unique prosthetic group 4-methylidene imidazol-5-one, which may indicate a similar
catalytic mechanism (Bode & Müller, 2003; Rother et al., 2001; Schwede et al., 1999). Due
to the fact that PAL is the first enzyme in the plant phenylpropanoid biosynthetic pathway, it
has received the greatest attention and is therefore the best studied member of this enzyme
family (Schuster & Retey, 1995). Since a PAL was not detected in the S. polyantibioticus SPRT
genome, an alternative pathway for the production of cinnamic acid was proposed whereby the
need for a PAL was bypassed (Chapter 3). This pathway was proposed on the basis of the
discovery of a cinnamate CoA ligase, encoded by SPR_60150, in the S. polyantibioticus SPRT
draft genome. SPR_60150 displayed significant homology to the enzyme, 4-coumarate-CoA
ligase (4CL, EC: 6.2.1.12), which has been characterized to occur in the biosynthetic route to
benzoyl-CoA in “S. maritmus” strain DSM 41777T. In addition, 4CL, which catalyzes the final
reaction of the phenylpropanoid pathway in plants, has also been characterized in S. coelicolor
A3(2) (Kaneko et al., 2003). Similarly to S. polyantibioticus SPRT, a PAL was not detected in
the S. coelicolor A3(2) genome. Therefore, if cinnamate or 4-coumarate are physiological
substrates, they may be supplied from dead plants in the environment, although no uptake
system for either of these compounds has been described in Streptomyces spp. (Kaneko et al.,
2003).
Three genes encoding a putative acetyl/propionyl-CoA carboxylase, a putative acyl-CoA
dehydrogenase and a putative enoyl-CoA hydratase exist in the immediate vicinity of the gene
encoding the 4CL enzyme in the genome of S. coelicolor A3(2) and, due to the fact that
functionally related genes are normally clustered in bacterial genomes, the authors speculated
that the 4CL, together with the enzymes encoded by the three other genes, are involved in the
Chapter 5 – General Discussion
230
production of a secondary metabolite (Kaneko et al., 2003). In light of this and the fact that a
putative enoyl-CoA hydratase and a putative acyl-CoA dehydrogenase were found next to the
gene encoding a cinnamate-CoA ligase in the S. polyantibioticus SPRT genome, this cluster
was identified as being putatively responsible for the biosynthesis of benzoic acid.
The proposed pathway involved the catabolism of phenylalanine to cinnamic acid via
phenylpyruvate and phenyllactate (Chapter 3). The D-lactate dehydrogenase encoded by
SPR_60260 and the cinnamate CoA ligase encoded by SPR_60150 were disrupted and S.
polyantibioticus single mutant strains lacking a functional copy of each of these genes were
able to produce DPO, thereby proving that they are not involved in the DPO biosynthetic
pathway.
Thus, benzoic acid does not appear to be synthesized in S. polyantibioticus SPRT via a PAL-
dependant pathway or via another cinnamate-containing pathway in which the PAL reaction is
bypassed. As studies have demonstrated that antibiotic production in actinomycetes and other
microorganisms makes use of amino acids as precursors, such as in the biosynthesis of
oleandomycin in S. antibioticus, the second approach to determining the genes responsible for
benzoic acid biosynthesis focussed on a novel variation of the phenylacetate degradation
pathway (Tang et al., 1994). Phenylacetic acid (PA) is a common intermediate in the microbial
metabolism of a variety of aromatic substrates including phenylalanine but, although there are
examples of aerobic, non-PAL pathways in nature (often utilising PA as the starter unit), none
of them is fully understood.
In the denitrifying bacterium, Thauera aromatica, PA is catabolized under anoxic conditions
to benzoyl-CoA via the intermediates phenylacetyl-CoA and phenylglyoxylate (Rhee & Fuchs,
1999). This mechanism is similar to the pathway described in A. evansii, in which it is
speculated that an aerobic pathway may coexist in order to fully metabolise PA. Moreover, an
aerobic pathway for the degradation of PA has been established in Gram-negative bacteria such
as Pseudomonas putida and E. coli, whereby PA is first converted to PA-CoA, which
subsequently undergoes ring hydroxylation, hydrolytic-ring opening and further degradation.
In contrast, this pathway and its role have not been comprehensively characterised in Gram-
positive bacteria, although a study of Rhodococcus sp. strain RHA1 provided conclusive
evidence that it does have a functional PA degradation pathway that is partially responsible for
Chapter 5 – General Discussion
231
the degradation of styrene, ethylbenzene and 3-hydroxyphenylacetate (Navarro-Llorens et al.,
2005). However, the existence of benzoic acid or benzoyl-CoA as a final product in these
aerobic pathways has yet to be established.
Furthermore, the PA catabolon has been described to be involved in the biosynthesis of the
seconday metabolite, antimycin A, produced by Streptomyces albus S4 (Seipke & Hutchings,
2013) and in the biosynthetic pathway for the production of neoantimycin, part of the antimycin
family of depsipeptides, by Streptoverticillium orinoci (Li et al., 2013).
In light of the fact that the PA catabolon has been associated with the production of secondary
metabolites and because a PA-CoA ligase is responsible for the catalysis of the first reaction in
this pathway, catalysing the activation of PA to PA-CoA, a homologue of the gene encoding
this enzyme, paaK, was amplified from the S. polyantibioticus SPRT genome. However, an S.
polyantibioticus mutant lacking a functional copy of this gene did not lose the ability to
synthesize DPO, thereby suggesting that a PA-CoA ligase is not involved in DPO biosynthesis.
However, due to the fact that six paralogous PA-CoA ligases (with an amino acid similarity of
24 %) were annotated in the S. polyantibioticus SPRT genome, it is plausible that one or more
of these enzymes may have suppressed the effect of the knockout of the paaK gene in S.
polyantibioticus strain ∆PAAK, which thereby resulted in a level of DPO production that was
comparable to the wild-type strain. Future work may necessitate the need to measure the exact
level of DPO production in both the S. polyantibioticus ∆PAAK and wild-type strains, as a
lower level of DPO production in S. polyantibioticus ∆PAAK could serve as circumstantial
evidence that a PA-CoA paralogue functions less efficiently than the dedicated enzyme in the
biosynthesis of DPO, thereby implicating the involvement of the enzyme encoded by
SPR_46390 in the production of DPO. Indeed, Geukens et al. (2006) demonstrated that Type
I signal peptidase (SPase) paralogous enzymes in S. lividans can only partly complement each
other, as clear differences in SPase substrate specificity resulted in a dramatic depletion of
preprotein processing and secretion.
The attempts made in this study to identify the genes responsible for the biosynthesis of benzoic
acid in S. polyantibioticus SPRT were based on characterised bacterial benzoic acid
biosynthetic pathways, as well as characterised bacterial amino acid degradation pathways.
Due to the fact that the in silico gene identification using a genome mining approach and
Chapter 5 – General Discussion
232
subsequent in vivo gene disruption experiments disproved both hypotheses on how benzoic
acid is synthesized in S. polyantibioticus SPRT, the genes encoding this pathway are still
unidentified.
However, since the shikimate and chorismate pathways (Figure 5.4) are commonly used to
generate molecules with benzene rings in bacteria (and other organisms), S. polyantibioticus
SPRT could perhaps use a novel variation on one of these aromatic biosynthetic pathways to
generate benzoic acid. Indeed, intermediates from the shikimate pathway serve as starting
points for the biosynthesis of secondary metabolites in bacteria and plants (Herrman & Weaver,
1999; Herrman, 1995). Eukaryotic benzoate biosynthesis also proceeds via phenylalanine
derived from the shikimate pathway and, most importantly, a study by Hoffmann & Grond
(2004) postulated a direct conversion of shikimic acid to benzoate in Streptomyces griseoviridis
strain Tü 3634. In S. griseoviridis strain Tü 3634, the hypothetical mechanism of benzoate
formation consists of the dephosphorylation of shikimic acid-3-phosphate and subsequent
dehydration steps involving the enzymes 3-dehydroquinate dehydratase and shikimate
dehydrogenase. Shikimate is converted to benzoate in an alternative microbial route that has
not been described before and was confirmed by 13C feeding experiments. This proved that the
unsubstituted benzoyl ring in the final product originates directly from shikimate, contrary to
the plant-like conversion of shikimate to benzoate via prephenate, phenylalanine and
cinnamate, in which the carboxylic carbon of shikimate is lost in the latter stages (Figure 5.5)
(Hoffmann & Grond, 2004). However, the enzymes that convert shikimate to benzoic acid in
S. griseoviridis strain Tü 3634 have yet to be characterized.
It is possible that S. polyantibioticus SPRT employs a similar pathway for the biosynthesis of
benzoic acid directly from shikimate. As would be expected for a central metabolic pathway,
the enzymes involved in the shikimate pathway are all present in the S. polyantibioticus SPRT
draft genome, namely 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase
encoded by SPR_10690 and SPR_58840, 3-dehydroquinate synthase encoded by SPR_41260,
3-dehydroquinate dehydratase encoded by SPR_41270 and shikimate dehydrogenase encoded
by SPR_41230. A suggestion for future work would be to disrupt the genes encoding enzymes
in the shikimate pathway (Figure 5.4) in S. polyantibioticus SPRT, followed by assays to
determine whether DPO is still produced. If an S. polyantibioticus SPRT shikimate mutant was
unable to produce DPO, it would imply that the shikimate pathway is required to provide a
Chapter 5 – General Discussion
233
precursor for DPO biosynthesis (i.e. benzoic acid). Once this had been established, further work
would be required to identify how benzoic acid is derived from shikimate (or one of its
precursors).
Another possible source of benzoic acid could be the mandelate metabolic pathway (Figure
5.6), which has been well characterized in Pseudomonas putida ATCC 12633 and allows
various pseudomonads to utilize mandelate as a sole carbon source via the oxidative
degradation of R-mandelate to benzoate (Tsou et al., 1990). Indeed the existence of
homologous enzymes from the P. putida ATCC 12633 mandelate catabolic pathway in the
draft S. polyantibioticus SPRT genome, including a mandelate racemase (encoded by
SPR_50530), a putative mandelate dehydrogenase (encoded by SPR_47860), a putative
benzoylformate decarboxylase (encoded by SPR_19760) and a putative benzaldehyde
dehydrogenase (encoded by SPR_9230) could confer on S. polyantibioticus SPRT the ability
to synthesize benzoic acid for DPO biosynthesis. However, due to the low degree of homology
shared between the S. polyantibioticus SPRT enzymes and the P. putida ATCC 12633 enzymes
involved in mandelate catabolism, and due to the fact that the genes for the enzymes identified
as putative mandelate pathway members in S. polyantibioticus SPRT are not clustered, it seems
unlikely that this method of benzoic acid synthesis is employed by S. polyantibioticus SPRT.
Another problem with any proposed mandelate-to-benzoate pathway is that an external source
of mandelate would be required and S. polyantibioticus SPRT is able to produce DPO in a
conventional bacterial growth medium from which mandelate is absent.
Chapter 5 – General Discussion
234
Figure 5.4 An illustration of the shikimate pathway found in microorganisms and plants. In a sequence of
seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to
chorismate, the precursor of the aromatic amino acids and many aromatic secondary
metabolites. All pathway intermediates can also be considered branch point compounds that
may serve as substrates for other metabolic pathways. The key enzymes involved in the
pathway are abbreviated in red: 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase
(DAHPS), 3-dehydroquinate synthase (DHQS), 3-dehydroquinate dehydratase (DHQD),
shikimate dehydrogenase (SD), shikimate kinase (SK), 5-enolpyruvylshikimate 3-phosphate
synthase (EPSPS) and chorismate synthase (CS). The direct conversion of shikimate to benzoic
acid, a branch point of the shikimate pathway, was postulated by Hoffman & Grond (2004) and
is catalysed by unknown enzymes.
Chapter 5 – General Discussion
235
Figure 5.5 A depiction of the biosynthetic pathway leading to the synthesis of benzoic acid directly from
shikimic acid in S. griseoviridis Tü 3634 and ruling out the plant-like pathway via phenylalanine
described by Hertweck & Moore (2000), as established by 13C labelling experiments (Hoffman
& Grond, 2004).
Chapter 5 – General Discussion
236
Figure 5.6 The P. putida ATCC 12633 mandelate metabolic pathway. Benzoate is subsequently converted
to acetyl-coA and succinyl-CoA by the enzymes of the β-ketoadipate pathway (Tsou et al., 1990).
Finally, benzoic acid biosynthesis in S. polyantibioticus SPRT may occur via the key
intermediate, chorismate. The chorismate pathway provides the aromatic building blocks for
secondary metabolites such as enterobactin, pyochelin and yersiniabactin (Van Lanen et al.,
2008). Chorismate can be converted to 4-hydroxybenzoic acid (4HBA) by the action of
chorismate lyase (Agarwal et al., 2014), of which three homologues (SPR_8160, SPR_40730
and SPR_74890) have been identified in the S. polyantibioticus SPRT draft genome.
Dehydroxylation of 4-hydroxybenzoic acid would lead to the formation of benzoic acid.
Indeed, the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA has been
Chapter 5 – General Discussion
237
reported under anaerobic conditions in Pseudomonas species (Glöckler et al., 1989). However,
there are currently no reports in the literature suggesting that 4HBA can be aerobically
metabolized via dehydroxylation to benzoic acid (Fairley et al., 2002).
5.4 Proposed pathway for the biosynthesis of DPO in S. polyantibioticus
SPRT
Most NPRS gene clusters abide by the so-called colinearity rule whereby each module is
responsible for one discrete chain elongation step and the specific order of the modules defines
the sequence of the incorporated amino acids (Wenzel & Müller, 2005). However, contrary to
the hypothesis that a linear NRPS system with the standard C-A-PCP domain order would be
identified for DPO biosynthesis in S. polyantibioticus SPRT, the cluster exhibited a nonlinear
arrangement with the core domains arranged as A-PCP-C, in addition to stand-alone A, C and
PCP domains. Initially, these nonlinear NRPS systems were assumed to be exceptions to the
colinearity rule, however, numerous examples of nonlinear NRPS systems have been reported
in the past few years, and it has become increasingly apparent that they constitute a
considerable portion of the range of NRPSs found in nature (Mootz et al., 2002).
Based on the genome annotation analysis and gene disruption studies, a model for DPO
biosynthesis can now be proposed. The source of benzoic acid is yet unknown, as the pathway
for benzoic acid biosynthesis in S. polyantibioticus SPRT has not been established, despite the
efforts of this study to identify it. The acyl-CoA synthetase encoded by gene SPR_52860 is
proposed to select and activate benzoic acid as a CoA-thioester in the first step of the DPO
biosynthetic pathway (Figure 5.7).
As the substrate specificity of the A domain encoded by gene SPR_53060 is unclear (as it could
bind either phenylalanine or 3-hydroxyphenylalanine), an in trans phenylalanine hydroxylation
step is proposed. Indeed, FAD-dependent monooxygenases encoded by any of SPR_48520,
SPR_49640, SPR_52630 and SPR_53180 could provide the hydroxylase activity to catalyse
the β-hydroxylation of a phenylalanine residue in DPO biosynthesis. The A domain, encoded
by gene SPR_53060, is proposed to be responsible for the selection and activation of
3-hydroxyphenylalanine through ATP hydrolysis. The β-hydroxyphenylalanyl-adenylate
would then be transferred to the PCP domain, also encoded by SPR_53060, and formation of
Chapter 5 – General Discussion
238
the amide bond with benzoyl-CoA would be catalysed by the lone C/Cy domain encoded by
SPR_53040. Heterocyclization of the intermediate, benozyl-β-hydroxyphenylalanine to 4-
carboxy 2,5-diphenyloxazole would then be catalysed by the putative Cy domain encoded by
SPR_53060 operating in tandem with the C/Cy domain encoded by SPR_53040. Indeed, the
antiSMASH analysis, in addition to a multiple sequence alignment with the corresponding A-
PCP-C NRPS module from S. ambofaciens ATCC 23877T, revealed the existence of a large
putative “recognition sequence” at the C terminus of the C domain (SPR_53040) and at the N
terminus of the A domain encoded by SPR_53060. In contrast to standard COM domains
which normally facilitate the interaction between PCP and C domains located on separate
multienzymes (Hahn & Stachelhaus, 2006; Hahn & Stachelhaus, 2004), intersubunit protein-
protein interactions in the DPO biosynthetic pathway may establish interfaces between the A
(SPR_53060) and C/Cy (SPR_53040) domains to provide a functional pathway. Thus, the
recognition sequence would mean that the C/Cy domain encoded by SPR_53040 would
potentially interact with the A domain encoded by SPR_53060, thereby forming a conventional
C-A-PCP arrangement.
The oxidation of the hydrolytically labile oxazoline to form the oxazole moiety in the
heterocyclization reaction is proposed to be catalysed in a manner homologous to the oxidation
process observed in pyrimidine biosynthesis.
Decarboxylation would be carried out in trans by a putative decarboxylase, such as the enzyme
encoded by gene SPR_68606, which shares 30% amino acid homology to the decarboxylase
found in the tautomycetin biosynthetic cluster from Streptomyces griseochromogenes. This
reaction is required to convert 4-carboxy 2,5-diphenyloxazole to DPO.
The type II thioesterase, encoded by gene SPR_53090, would catalyse the release of DPO by
breaking the covalent linkage between DPO and the 4’-phosphopantetheine (4’-PP) thiol arm.
Even though the inactivation of the gene encoding this enzyme within the S. polyantibioticus
SPRT genome did not abolish DPO biosynthesis, the stand-alone type II TEs, often encoded
within NRPS gene clusters, do not play an essential role in NRP synthesis because their
removal from NRPS systems has been shown to decrease product titres, but not completely
eliminate peptide synthesis (Schneider & Marahiel, 1998).
Chapter 5 – General Discussion
239
Finally, two NRPS domains found within the putative DPO biosynthetic cluster are believed
to be inactive. The PCP domain encoded by SPR_53070 is postulated to be inactive due to the
absence of the critical catalytic serine residue found in the conserved signature motifs of known
PCP domains (Chapter 3; Figure 3.7) and the C domain encoded by SPR_52900 is believed to
be inactive due to the absence of the H-H-x-x-x-D-G motif found in all active C domains.
Figure 5.7 Proposed DPO biosynthetic pathway in S. polyantibioticus SPRT.
Chapter 5 – General Discussion
240
5.5 Conclusion and future work A gene cluster responsible for the biosynthesis of DPO was identified in this study and, based
on the genome annotation analysis and gene disruption experiments, a model for DPO
biosynthesis is proposed. At this stage, the model cannot account for the source of benzoic
acid, as in vivo gene disruption experiments disproved both of the hypotheses on how benzoic
acid is synthesized in S. polyantibioticus SPRT. However, alternative hypotheses regarding
benzoic acid biosynthesis in S. polyantibioticus SPRT have been put forward and are suggested
as the place to start in future studies to elucidate the production of this unusual starter unit in
DPO biosynthesis. The heterologous expression of the putative DPO gene cluster, identified
in this study, in host organisms such as ‘Streptomyces maritimus‘strain DSM 41777T, where
benzoic acid biosynthesis occurs, could be used as a method to confirm the involvement of
benzoic acid in DPO biosynthesis.
Future work should also involve the characterization of the substrate selectivity of the A
domains encoded by SPR_52860 and SPR_53060, in order to confirm their specificity towards
benzoic acid and 3-hydroxyphenylalanine, respectively, using an ATP-PPi exchange assay.
Additional liquid chromatography-mass spectrometry (LC-MS) analyses of the antibacterial
compounds produced by both the wild-type S. polyantibioticus strain SPRT and constructed
disruption mutants would confirm the identity of the bioactive compounds and further
corroborate the results obtained from HPLC and TLC-bioautography analyses, thereby
strengthening the preliminary hypothesis pertaining to DPO biosynthesis.
Additionally, DPO biosynthesis is most likely controlled by SPR_52890, the only gene in the
putative DPO biosynthetic cluster predicted to be involved in regulation. This gene encodes a
putative transcriptional regulator that contains a C-terminal DNA binding HTH domain and
belongs to the two-component response regulator family. In order to identify the gene cluster
responsible for the biosynthesis of benzoic acid in S. polyantibioticus SPRT, the presence of
cluster situated regulatory genes, such as SPR_52890, and/or promoter sequences can be used
as a tool to search for any interconnected pathways in the genome.
Moreover, this study was successful in the optimization of a transformation method for the
introduction of DNA into S. polyantibioticus SPRT. In light of this, future work should entail
Chapter 5 – General Discussion
241
gene disruption experiments in order to determine the involvement of the genes postulated to
be inactive, such as the PCP domain encoded by SPR_53070 and the C domain encoded by
SPR_52900. In addition, gene disruption of the genes identified in Chapter 3 as having an
unknown function in DPO biosynthesis will help to define the boundaries of the DPO gene
cluster.
Furthermore, the identification of the gene cluster responsible for DPO biosynthesis has laid
the foundation for combinatorial biosynthetic studies to create derivatives of DPO that might
be used in the treatment of drug-resistant tuberculosis. Lastly, the S. polyantibioticus SPRT
genome sequence could be explored further to identify the antibiotic gene clusters for other
potential antitubercular antibiotics that this organism produces.
Chapter 5 – General Discussion
242
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Appendix A
247
APPENDIX A
Figure A1. Genome map of the draft S. polyantibioticus SPRT genome generated using the CLC Genomics
Workbench 7.0.4 (CLCbio, Denmark). Arrows represent the relative positions of the secondary metabolite clusters discussed in Chapter 3, where (A) is the NRPS gene cluster spanning the region located from nucleotide 637827-688770, (B) is the NRPS gene cluster spanning the region located from nucleotide 669778-735243, (C) is the NRPS gene cluster spanning the region located from nucleotide 3093585-3154158, (D) is the NRPS gene cluster spanning the region located from nucleotide 3320540-3386004, (E) is the NRPS gene cluster spanning the region located from nucleotide 5343994-5398013 and (F) is the NRPS gene cluster spanning the region located from nucleotide 5634984-566500.
Appendix B
248
APPENDIX B
Nucleotide sequences of the genes preliminarily identified as components of the DPO
biosynthetic gene cluster are provided in this appendix.
> SPR_52860
ATGCCCTCGACCACTGAATCCGCCGCCACGCTGTGGGAGCTCATCGACCGGGCCGTACGCCTCTCCCCCGCGGCC
ACCGCCGTACGCCAGGGAACGCGGGCCCTCACCTTCCGCGAACTCGCCGACCGCGTCGAGACCACAGCACGCCGC
CTGCGCACGCGCCTGCCCGCCGACGGCGGAGGAACCGTGGGCCTGCTCTTCGAGAACACCGTGGAGAGCACGGTG
GCCTTCCTGGGCGCGCTGTACGCGGGCGTACCCCTCACGCCCCTCGAACCCGACAGCACCGAGCCCCATCTCCTG
GGCGTGCACCGGGACTTGGGACCCCTGCACCTCGTCGGTCGGCAGGCGAGGCTCAGCACACTGGCCCCTCCCGCT
GCCACCTCACTCGCCGCCCGGTGGCAGGGCGGTGTCCTCATCGACGTGGACGAGCTCACCGCCCGCCCCGGCGCG
GCGGCCCCGTCGGCGCCGCTGCCCGCGCCGCCGCCCGACGCCCCCGCGCTCTACCAGTACACCTCCGGCTCGACC
GGCGAGCCGAGGGCCGCCGTGCACTCACAACACGACCTGGTCCGCGGTGGCGAGATCTACGCCCGCACCTACGGC
ATCACCCCGGCCGACCGGATTCTCGCGGCCGTACCCCTGCTGCACTCCTTCGGCATGGTCGCCGCGCTCGCCACC
GCGCTGCACGCCCGCGCGGAGCTCGTCCTGCTCGGCCGGTTCGCACCGGCCGAAATGCTCCGGGCACTGCACCAG
CACGCCTGCACCATCGTGGTGGGCACCCCGCTCGCCTATGACCTGGCGGCCCGTTCGGCGGCCTCGCGCAGCGAG
ACGTCCCGCCCGGGCGACACCGTACGGCTCTGTCTGTCCTCCGGGGCGGCCCTGCCCCCGGCCGTGGCGGACCGC
TTCGCCCAGCACTGCGGCCCGGCCGTCCAGCAGGTCTACGGAAGCACCGAGGCCGGTGTCGTCGCCGCGCAGCTC
CCGCAGCCGGACGGCACGGCCGATGCCGGGGTGGGCAGGCCGGTGCCCGGGGTACGGATCCGCCTGGTGGACGAG
GAGGACCGACCGGTTCCGCCGGGCGGCACGGGCGCCCTGCTCGTGCGCACCCCCGCCATGTTCACCCACTACCTC
GGCCACCCGGGCCCGTCCGAACGAGCCTTCCGCGACGGCTGGTACGTCACCGGCGACCTGGCGCGCCTCGACGGT
GACGGCCGTCTGCACCTGGTGGGCCGCAAGGAGTCCTTCATCAACGTCGGTGGCAAGAAGGTCAATCCCCTGGAG
GTCGAAGCGGCCCTGCTGGCCCACCCGGCCGTCGCCGAGGCCGTGGTCTGGGGCGAGGTCGTGGAGGAACAGACC
AGCGAGCGCGTACGGGCAGCCGTGGTGCCGTGCTCCCCGCTCAGCGGGGCCGCGATCACCGCGCACTGCCGTGAC
CGGCTCCTGGCCCATCAGGTGCCCGCGACGATCGAGTTCGTGGCGTCGCTGCCCAAGACCTCCCTGGGCAAGATC
CGCCGCGCGGCCGTGGCCGGAGCCGCCCCCGCACGGCACCGGGACGACACGGCCTGA
> SPR_52870
ATGGCACTTCTGGACGGCATCGAGGCGGGCGCCGAGGACGCCTACGACCGCCATTACGGCGACCGGCAGCTGCTG
CGCCGCATCACCCCCTACTTCCAGGGCCGCGGCGGCGCGGTGGCCTTCGTCACCGCGGCGGTGGTCGTGGCGGCG
GCCGCGGGCAGCGCCGTACCGATCCTGCTGGCCCGCACCGTCGACTCGGCCCTGGACGACGACACCCGGTCGGCG
ATGACACTGCTAGCCGTTGCCGTCCTCCTCACCGGCGCCGCGGCCTGGCTGTTCACCGTCCTCCAGCAGCGCCTC
ACCGGCACCCTTGTCGGCGACGCGGTGCTGCAACTGCGCGAACACGCCTTCGCCGCGGCCGTACGCCAGGACCTC
GCGTTCTACGACACCCACCCCACGGGCCTGGTGGTCAGCCGCGTCACCAACGACACCCAGACGTTCGGTGCCCTG
CTCTCCCTGGTGCTCTCCCTCATCGGTCAGGTCCTCGTCGTCCTGCTGATCCTGACGGCACTCTTCGTCATCAAC
GTGCCGCTGGCGCTGCTCACCACCGTGGTGGTCGCCCTGATCGTCGCGGTCAGCCTCGCCTTCAGGAAACGCGCG
CGCACCGCCTCCACCCGCCAGCAGCAGGCCCTCGCCGAGGTCAGCGGCTATGTGCAGGAGACCCTGCGCGGGATC
ACGGTGGCCCGCAACCACCGCGGCGAATCGGCGGCCGAAGCGGGGCTGCGGGCGGTCAACGGCCGCTGGTACCAG
GCCAGTGTGCGCCTCAACCGCCTCTTCAGCGGCATCTTCCCGCTGCTCATCTCCCTGACCGGACTGGGCACGACG
GCGGTGGTGCTGGCCGGCGGCCACCAGGTCGCATCCGGCGACATCTCGGCCGGCGAATGGGTCCTCTTCCTGGAG
GCCCTGGCCCTCTTCTGGTCGCCGGTCGCCACCATCGCCTCCTTCTGGCAGCAGCTCCAGCAGGGCCTCTCGGCC
GGGGAGCGGATCTTCGCGCTGATCGACCGGGAACCCGCCGTCCACCAGAGCGCCCAGCTGCCCCCGCCCCGTCTG
CGCGGCGCACTCGAACTGCGCGGCGTCAGCTTCCGCTACACCCCCGAGCGGCCCGTCCTCCACGACATCGACCTC
ACCGTCGCGGCCGGGGAGACCGTCGCACTGGTCGGCCACACAGGGGCGGGCAAGTCCAGCCTGGTGCGCCTGCTG
ACCCGCTCGTACGAGTTCCAGGAAGGCAGCCTGCTCGCCGACGGCCGGGACGTGCGGACCTTCGACCTGGCGCAG
TACCGGCGCTGCCTCGGCGTGGTCACCCAGACGCCCTACCTCTTCTCCGGCACCGTCGCCGAGAACATCGCGCTG
GGCCGGCCGGGCGCCGACCGCGCGGCCATCGAGGCCGCGGCCCACGCGGTCGCCTCCGGCGCCTGGCTGCGGCAG
Appendix B
249
CTGCCCGACGGCCTCGACACACCGACCGACGAAGGCGGCCGCAACCTGTCGACCGGGCAGCGGCAGATCGTGGCG
CTGGCCCGGGTGTTCCTGCAGGACGCCCCGATCTTCATCCTCGACGAGGCCACCGCCAGCGTCGACCCGCTCACC
GAGGCCCAGATCCAGGAAGGACTCGACGCGCTCGCGGCGGGGCGCACCACCATCGTCGTGGCGCACCGGCTCCCC
ACCGTCCGCAAGGCCGACCGCATCGTCGTCGTCGACGACGGGCGCCTCGTCGAACAGGGCGACCACGCCACCCTG
ATGCGGCAAGGCGGTGCCTACAGCCGCCTCTACCGGCAGTACTTCCACCATCAGCACCCCGACCACGACCCCGCC
GACGACCCGTCCGGCACCGGGCGGGCGCCGGTCGGCGCGGGGCCGCACCTAGCGGAGGCCAGCGATGCCCTCGAC
CACTGA
> SPR_52880
ATGACGACCGAGACCGCCGCGCAGACCCGCTCCGCGTCCGGACCGGCGCCGGCCACCGGGCCCGAACTGACCATC
GGCCACTGGCTCCGCCTGCACATGCGTCACTTCCGCCGGCACATCGCGGTCCATCTGACCGGAAGCCTGCTCTGG
CAGTCCCTCACCGCGGCCCTCCCCCTGGTCATCGGGCTGGCCTTCGACGCCGTACTGAAGACCAACGGGCGGGAA
CCTGATCTGGGGGCCCTGCACGCCGTGTGCGCGGCACTGCTCGCCCTGGTCCTCGTCCGGGGCGTCTGCGGCATC
GCCTCCACCTACGCCCTGGAGGCCTTCGCCAGCGGCCTTGAACGCGACGCGCGGGCCGGCGTGTTCGCCAGCATG
CTCCGCAAGAAGCAGGGCTTCTTCAACCGGCACCGCACCGGCGACCTGTCCACCCGCGCCACCGGCGACGCCGAG
GCACTCGGACTCATGGTGTCGCCGGGCTTCGACATGACCCTCGACATGGCGCTCAACACCCTCATTCCGCTGGTG
TTCATCGCGGCCGTCGACTGGCGGCTCCTGCTCTTCCCCGGGATCTTCGTCGTCCTGTTCGTCGCCGCCCTCATG
GATCACGGGCGGCGCCTCGAACCGGTCTCCGACCTCACCCGCGAGGAGTTCGGCGCGATGTCCGCCCAGGCCGCC
GAGAGCATCACCGGAATCGAGGCGGTCGAGGCGACCGACGGAGCGGAGCGCGAGCAGGACCGCTTCCGCCGCACC
GCCACCGCCTACCGGGACGCCGCCGTGGCGCAGGCACGCGTCCAAGCCCTGTCCTTCCCCCCGCTGCTGCTCGCC
GTGGCCACCGCCGGAACGCTGCTCCACGCCGTCTTCCTGCTGCGCGACGGCTCACTGAGCGTCGGTGAACTCGTC
GCCGTCCTGGGCCTGATGAGCACGCTGCGCGCCCCCACCCAGCTCGCCTCCTTCAGCATCGGGCTCATCTTCTTC
GGACTCTCCGGCGCACAGCGCATCCTGGAGATCATCAACGACCGGGCGGGAGAGGACGAGGAGGGAGGCCGGCAC
GAGGCGGTCATCACCGGCGAAGTGGTCTTCGAGAACGTCACCTTCGCCTACGGAAACGGCAAGCCCGTGCTGCGC
GACATCTCCTTCCGCGCCGCCCCCGGCACCACCGTCGCCGTCGTCGGCGCCACCGGCAGCGGCAAGTCCACCCTG
CTGCACCTGCTGAACCGCACCTACGTGCCCACCGAGGGCCGCGTCCTCGTCGACGACGTGGCCACCACCGCCTGG
GACCCCTCGGCACTGCGCGGCCAAGTAGCCGTCGTCGAGCAGGACGTGGTCCTGTTCTCCCGCACCATCGCCGAG
AACCTCGCCTTCGGCGCCGCACCGGACACCGGCCGGGCCCAACTGGAAGAGGCGGCCCGTACCGCCCAGGCGCAC
GACTTCGTCATGGCGAGCGAGCACGGCTACGACACCGTGCTGGGCGAGCGCGGCGTCACCCTCTCCGGCGGCCAG
CGCCAACGCCTCGCCATCGCCCGGGCGTTGGTGACCGACCCGCGCATCCTCGCCGTGGACGACGCGACCAGCGCC
GTCGACAGCGTGACCGAACACGAACTGCAGCTCGCCATGCGGCGCGCCGCCACGGGCCGTACCACCTTCCTCGTC
ACCCCCCGGCTGTCCCGCATCCGCGCCGCCGACCACATCGTCGTCCTCGACGCGGGCCGGGTCGTCGGCCAGGGC
ACCCACGAACACCTCCTGCGCACCTGCGCGTTCTACCGCACGATCTTCGCGCCGTACGCACTGCCGGCCGCGGAG
GGAACCACCACCGCCGCCGTCGCGGCCGGGGCAGAGGAAGGAACCCGCTGA
> SPR_52890
GTGAGCGATTTAGCATGCAAACCCCCCGCCCCGTCGCGACCGCTGGTCGTCGCGACTCTTGTCCACGACGGCCTC
GCGCGCCAGGAGCTGCAGACCGCTCTGGCACAGGCCGCCCTCGTGCGCGAAGCCCGGCACTGCGCCGACCGACGC
GAGGCGCGGGAGGCTCTGGACTCCGGCCGGGTCGACGTGTTCGTCGTGGCCAGGAGCGACTACGACGCCCATGCC
GACTTGCTTGACGACTTCCGCTCGCCCCTGCCGAGGATGCTTGTGCTGTTGAGTCATGCCGATCCGGACGAAGCG
ATGCTGTCCGGGCCACCAGCGCCCGATGGATTCCTGCTGCAGGGCGGTCTGAGCGCCGCCGATGTGGACAGCGCG
CTGCGGCGCATGGCCGCGGGCGAGGTACCGATGCCGGCCCTGCTCGCCCGCGCCCTGATGGACCGTGCCAGCACG
AGCCGCCTGCCGCACGGCCGACATGACATCGCTGTCACCGCCCGGGAGACCGAGGCGCTGCTCCTGCTCGCCGAG
GGGCTGAGCAACAAGCAGATGGCCCGGCGGCTGGAGATCACCAGCCATGGCGTGAAACGGCTCGTGGCCAGTCTG
CTGCTCAAGCTCGGGGCGCCGAACCGGACGGCTGCCGTGGTCCTGGCCATCCACGCGGGTCTGATCCGCACGACC
CGGCCGGGCCAGGGCACGGCGGACCGCCGGGTGGTACGGGTCCAGCTCGCGGGGGGTGGAGCGGACGTCCGCCTC
CCGGTCCCCGCCGTGCGCTTCCTCGGTGAGGAGGTGCCCGAGGAGCGCGCCTCCTGA
> SPR_52900
GTGGAAAAAATCGAGACGCGTGATGTCCCCTTCTACGGTCGCCGTTCGGTCATGGCGTCGGCCACCTGTGCACAG
CGGCAGATGTGGGACCTGATCCGACGGGAAATTCCCGACGGCTCCTTCTACGATTTCTGCCACGAGGTTTCCCTT
ACCGGCGAAGGAACCGTGGATGACGTTCTCGCCGTATGCGCGGAACTCCTCTCCCGGCATGAGTCGCTGCGTACG
GCATTTTTCTGCGGTGCGGACGGGAAATTGGTGCAGAGCGTGGCGCGGTCCGGATCCTTCGAGGCGGAGATATGC
GCCACCGGGCCCGGCGAGAGCGCGCAATTCGTGTACGACGCCTGGCGGCGGCGGATGCGGGACAGGACGTTCGAC
CTGCGGAACGGCCCGCCGCTGCGGGCGCTGGTCGTCGTCACCGGGCACGCGCCCGTCATGGCGGCCTTCTGTGTC
TCGCACCTGGCCGCGGACCTCATGTCGATGCGCACGCTGGCCGGTGAAGTCCTGCCCCTGCTGGCCGCGCGGATC
GCCCGTASCCCCGCCCCGCCGCCCGCCGTGACGCGCCAGCCGGTCGAACAGGCCGACTTCGAGCACTCCCCACAG
GGGCGCGCCCTGCTGGCCCGGGCCCACACACACTGGCGGCAGCAGCTGGCGAGCGCCCCCACCACGATGTTCCCC
Appendix B
250
GGCCGTCCCGCCCCGCAGGGGCCCGGCCGACCGGACCGCTACAGCGCGGCCATGGACTCCGCGGCCGCCTTCCTT
GCCATCCGTGCCCTGGCCGTCCGGTGGCGGGTGAGCACCTCCGCGGTGCTCCTCACAGCGGTCGCACTGCTGCTC
GGCGCCCGCTCCGGGCAACAGACCTGCGCCCTGAGGCTGTTGGCCGCCAACCGCACGCACCCCGCACTCCAGCGA
AGCGTGGCCAACCTGCACCAGGAGGTCCTCACCAGCTTCGACCTGCGCGGGGAGAACGTGCGGGCCGTCGCCCGC
CGCGCCTTCGCCGCGGGGACCCTCGCGTACGCCAACGGCCTGTTCGACCCCGACGGGGCGAACGAGCTGATCCGG
GCCGAAGGGCGCAGGAGGGGCGAGCCGCTCCAACTGTCCTGCTGCTTCAACGACATCCGCACCGACCACGATCCG
CGGAGTCCGGGCGGGACGGCGTCCGCCGAGCAGATCCGCGCCGCCCTGGCCCGTACCGTCGTCGCCTCCAGCGAC
TTCGAGGAAGCGGAGACCTTCTTCCTGGTCGTCGTCGACACCGACCCCGGGTGGCTCAGGTTCGTGCTGTGCGCC
GAGACGGCCGCCCTGTCGCCCGAGGAGGTGCACATCTTCCTCCGCGACCTCGAACGGCTCCTCGTGGACTGCGCC
GAGCAGCCCGAACGTTCCTGGCCACGACTCGATCAGGGCCGGGGCCCGCAGGCCGCCCAGCCACCGCGGACGGCT
GCCCGGGGATGA
> SPR_52910
ATGCCGCAGCACCGGGTGACGGGGGAGTCGACTGTGTTCACGGAAGCATTGGCACGTGAGGCTGCCATGGACGTG
GAGAACCCCGCGCACGGCACGGTGTTCGCGCAGGCTCCCCGCTGCACCGCCGCCGAACTCGACGCGGTCCTCGCC
GCCTCGGCCCGGGCGTTCCCCGGGTGGGCGGCACTGCCGCTGGAGAGCCGGCGTCCGTACCTGCTCGCCTGCCGG
GACGCGCTGCGCGACGCCGAGGACGACGTCGCGGACCTGCTGACCCGGGAACAGGGCAAGCCGCTGCGGCACGCG
CACCACGAAGTGCGGCTGGCCGCCGACTGGTTCGCGCACACCGCCGAACTCGCCCTGCACGCCGACCGGATCGTG
GACGAGCCGACGGCCCGCGTCACCCTGGAGCGGGTGCCGCACGGTGTCGTCGCGGCGATCGCCCCCTCCAACTAC
CCCGTCCTGCTGGCCGTGTGCAAGATCGCCCCGGCGCTCCTGGCGGGCAACACGGTCGTCCTCAAGCCCTCGCCC
GCCACCCCGCTGTCCAGCCTGCTCATGGGCGAGGTGCTGGGACGGGTCCTGCCGCCCGACGTCCTGCGGGTGATC
AGCGGCGACGCCGCGCTGGGGGCCCGGCTCACCGCACACCCGGCAGTGCGGCTGATCTCCTTCACCGGCTCGATC
GCCGCCGGCCGCGCCATCGCCCGGTCCGCCGCCGCGGACTTCAAACGGACCGTCCTGGAGCTGGGCGGCAACGAC
CCGGCCATCGTGCTGCCCGGCGCCGACGCACAGGCCCTGGCGCGTCCGCTGTTCGACCGGGCGATGGTCAACAGC
GGCCAGTTCTGCGCCGCGGTCAAGCGCGTCTACGTGCCGCGAGCCCAGCACGCCAAGCTCACCGCGGCCCTGGCC
GCGCCGGCCGAGGCCACCGTGGTCGGTGACGGCCTCGACGCCGCGACCGAACTCGGCCCGCTGGTCAGCCGTGAG
CAGCTCGCCCATGTGACCGCGCTGGTGGGCGACGCGGTGCGGCGGGGCGCCCGGCTCGTCACCGGGGGCCGCGCC
CTGGAGCGGCCCGGCCACTTCTATCCCCCCACCGTCGTCACGGACTTGCCGCAGGGGACGCGGCTTGAGGAGGAG
GAACAGTTCGGCCCCGTGATCCCCGTGATCGCGTACGACGATCTCGACGCCGTGACGGCCCGCGTCAACGCGTCC
CCCTACGGGCTGGGTTGCTCCCTGTGGGGCGATCCGGAACAGGCCGCCGCGGCGGCCCGGCGGCTGGACTGCGGA
ACGGTGTGGATCAACACCCACGGCGACCTCAGACACGACGTGCCGTTCGGCGGCCACCGGCACTCGGGCACGGGC
GTCGAGTACGGAGCGTGGGGCCTCCTGGAATACACCCAGATCACCATCCACCACCTCGCGCTGCGCGGGGGAGAT
GAGGGGAAGGCATGA
> SPR_52920
ATGAGCGAAACCACCTCCGGCGGCGCCCCCGGCACGAGCCTGGTCGATCTGTCGGGGCTGCGCGTGTTCGTCGGC
GGCCCCATCCAGTACGCCCTCGGGCACGACAGCTTCCACCCACCGCTGCGCGACACGATCCAGGCCATCGTCACG
GCGGTGACCGAGGCGGGGGCCACCGTCTTCTCCGCCCATGTCGTCGAGAGGTTCGGCCGGGACACACCGCACTTC
TCGCCGGAGGACGTCAGCGCACGGGACCTGGACTGGATGCGCCGGTGCGACGTGTTCGTGCCCGTCCTGCCGGCG
GACGACGGTGGCGGAGTGATGCGCACCGACGGCACGCACATCGAGATGGGCTGGGCGTCGGCGCTGGGCCGTCCC
ATCGTCATGGTGACCCCCCTGCCGGTCCCGGCCGGGGCCAGCCATCTGCTGCGCGGGCTGCCGTCCGTGGCGGAC
GTCGGCGGCGTCGACCTGGCCGAGCTGCGCCGCGACGGCCCCGGTGAACTGCTGCTGCGGCTGGGCAAGGTCGGC
CGCGACGCGGTGGTGACACCATGA
> SPR_52930
MTTVSHAASATEVLGLRLVSPLVVGSGLLTDQERNIRRLLTGGAGAVVTKTIHPGPLPAGDERLLHLPTGMINST
TYSRRGVDSWCATLSRFADDDLPVIASVHADSPAALADLAARVADTGCRALELGISCLNEDGGLADSARRVADYT
GAVRRATPLPFSVKLAVGEQVGERVSAAVEAGADAITLSDTIAGLAVSPDTGEVLLGRPFGYSGAGIKPLVLAAI
YELRRGGLSVPVMGSGGVRSGTDVAEYLTVGADAVQVYTALHTHMHQTLAEIRGGFDTWLGARGGSVADVVGRAL
DGGRGEVRAAADHERHGLR
> SPR_52940
GTGAGGTGCGTGCGGCAGCGGACCACGAACGTCACGGCTTACGGTGATCTGACCAGTCCCGAGACACCCGCGGCG
CTGAGCGGCGCCACTCTGGTCTGGCCGGTGGGCGGCCTTGAGCAGCACGGCCCGCACCTGCCCCTGTCGGTGGAC
TACGACATTCCCGACGCGCTCGCCCGGCAGCTGGTGGCGGACGTCGACGGCGTGCTCTTCCCGGGCCAGCCGCTG
TCCGCCAGGTCACTGCCGCACAGCGGCGGCGGACTGCGCTTTCCCGGCACCGTCCACATCGACGGGGGAACCTTC
ATCGACTACGTCGCCCAGTGCCTGCGGGCACTGGGCAGGCTCGCTCCCGCCCGGCTGGTGGTCGTCAACGGGCAC
Appendix B
251
TACGAGAACGAGGCCCTGCTGTTCGAGGCGATCGACGGACTCGACCCGGCGCGCACCTTCCCCGCCACCGAGATC
GTCGCCTTCAGCTGGTGGAGCCTGGTGGAGGAGGACTGGCTGCGCAAGCGGCTGCCCGAATTCCCGGGCTGGCAC
GCCGAGCACGCCGGGGTGACCGAGACCAGTCTCATGATGTACCTGCGTCCCGAGGTGGTCCGCCCGGTGCGGGTG
GACCACCCCACTCCGCCCGCCGCCGGTGTGTACCGCCACCCCGTCGACGCGGCCCGCATGTCCACCCAGGGGGTC
CTCACCACGACCTCCGGTGCCAGCGGCGAGCTGGGGGAGGAGCTGTTCTGGCACGTGATGGACGGCGTGGCACGG
ACCCTCGCGGACAGCCCGTCCCCGCGCACCGAGAAGCGCGCAAGCGAGTGA
> SPR_52950
ATGCGCCCGCCGACGACCGAACGCGACGACTTCGAAGCCCGGCTGCAAAGCCTCTTCGGCGACAAACTCCGCCCC
GTGGAGGAGAGTTTCGAGGCCATCCAGCACTTCAAGGACGGTTCCTTCGCGGTGGGGCAGCTCGGCCTCATGCTC
TACACCAACGGATACAACCTGCGGGGCACCAGCACCCGTCATCCGAACGGAATGGTCTTCCACGACGGGCAGAAC
CTCTTCGGCGTAGGCTACTTCAGCAAGGAACAGGACGAGCGCAAGCACCTGCACATCGTCGCTCCCAAGGGCAAG
GACCGGGTCGCCGCGGTCAGGTCGTTCATCTCCGCGACCCGCGAGGCGGGCCTGGCACACACCTCCGTCTACGTA
CGCCACCTCTCGCCGGACGACCACGCGCTGTTCCTGGCGGCCGGCTTCGAGCCGGTGGCGGCGGACCCCTGGCAC
CCCGAGGCGCCGGAAGAGGACGAGACCTACCCCAACCGCGTCTACCGCCTGGACGATCTGCTGGCGGTGGACGCC
GACGGCCGCCTGGTGGTGAAGAACCTGGCAGGGGACGGCAACCGGCGTCACAAGAACAAGAACCGGCTCGCCTAC
CGGCGTTTTGAGAACTTCCTCGCCCGCAACGACCACCTGGAGCTCCGCATCCGTCCCTACGGATACGGCCCCGAC
GAGGCGAAGATGGCCAGGGGCGTGGTCGAGGGCTACTTCGAAGCCCGCCGGGCACAGGGCGAGGTCGTCGGCTCC
ACCCCCGAGGACTACACGGCGATCGTCACCCAGCGGCCCGGAGGCCGCAACGAACACGACTACTTCGCCTACCTC
GGCGTCCTGGCCCAGCAGGGCGGCGAGGAGGTGCCCGTGATGTTCTTCGCGGGCGAGCGCACCGCGCCGCACAGG
GCATCGCTGTACTGCACGATGTCGATGCGCTTCGCGGACCGTATGAGCGGCCTGTTCAAGGACGCCACGGGATTC
ACCGCGATTCCGCAGTACATCTGGCTGACCGTGTTCAAGAAACTGTGGGACCGCGGCATCCGTGAGGTCGACGCG
GGCGGATCCGAGGTCAAAGGCCTCGACGACCAGAAACGACAGTTGGGCGGACGGCCCGAAAAGACCCACTGGGTC
GTGGGCTGA
> SPR_52960
ATGCAATCGCGCACCGCGCACCAGGACTTCACACCGTTCGACGTCTGCGTCGTGGGGATGGGATACGTCGGCGTC
ACCCTCGCCGCCGCCCTCCTGTCCACCGGCAAGCGGGTGCTGGGCTACGAGAGCGACCCGGCCGTCGCGGGCGAC
CTGGCACAAGGCCGCCTGAGGCTGGCGGAACCCGGCGTCGCCGAGCTCATCGAGCGGGGAGCCGCCGACGGCACG
CTCGCGGTCACGGCCGACATCTGCGGGCACCGTCTGCCACCCGTCGTCGTCATCTGCGTCGGCACACCCATCGCG
CCGGGCGGCACGACGCCCGAACTCGGCCATCTGTCGGCCGCCGCCGAGGCGGTCGCCGCGGGAGCGGACGAGAAC
ACCCTGGTGATCGTGCGCAGCACCGTCCCCGTGGGCACCACCCGCGAACTGGTCCTGCCGGCCCTGGCCCGCCGC
GTTCCCCAGCCGCTGCTCGCCTTCTGCCCCGAGCGCACCATCCAGGGCAAGGCACTGGCGGAACTGCTGTCCCTG
CCGCAGATCGTCGGCGGTCTGACCGAAGAGGCAACGAAGCTGGCCGCGGGGTTCTTCACCACCGTCAGCGGGCGC
GTCGTCCCCGTATCCACACTGGAGGCCGCCGAGCTGGTCAAACTGGTCAACAACTGTCACACCGACCTCATCTAC
GGCTTCGGCAACGAAGTGGCCCTCATCGCCGAGAAGCTCGGCCTGGACGCCATGGAGGTCATCACCTCCGCCAAC
ACGGACTATCCGCGCCCCGATCTGAGCAGGCCCGGCTTCGTCGGCGGCAGCTGCCTCACCAAGGACCCCCACCTC
CTCGCCTACTCCCTCGCCCGCCACGACCACACCCCGCAGATGGTCATGGCGGCCCGCACCCTCAACGAGTCCATG
CCCCGCAGGGTCGGCGAGCGCGTGCTCGACGCGCTGCGCCGGGACGGCCAGGACCCGCAGCGGTCCACCGTCCTC
GTCTCCGGATTCGCCTACAAGGGCCGGCCCGAGACGGACGACCTGCGCGGCGCCCCCTGTGTGCCGCTGCTGGAG
TTCCTGCGCGGCAAGGTCGCCCGGGTGGTCGGGCACGACTTCGTGATCCCGCCCGAACGCATCGCGTCACTGGGC
GTACACCCCGTCACCCTCACCGAGGGGTTCACCGGCGCCCACGCCGCCATCCTGCTCAACGACCACGCCCGGTAC
GGCGAGCTGCCGGCCGACGAGCTCATCGCCCGGATGAGCCCGCCGGCGCTGGTGTACGACGCCTGGCGGGTGCTT
CCGCAGACGACGAAGACAATGAGGCTCGGCAGTGCCTGA
> SPR_52970
GTGCCTGAGAAGATCCTGGTCACCGGGGGAGCGGGGTTCATCGGGCTGCATCTGGCCGCGGAACTCGCCTCCCAC
CACGACGTCACCCTCCTCGACGACTTCAGCAGGGGCCGGCGCGACGCGTTGCTCGACTCCCTGCTCGACCGTGTC
ACGCTCGTCGAGCACGACCTGACCACCCCGATTCCCGACACCCTTCTGCCCGACGACTTCGACACCGTTCACCAT
CTGGCGGCCGTCGTCGGCGTAGTCCACTCCAACGAGGAACCGCAGCGGGTGCTGCGGACCAACCTTCTGTCCACC
GTGCACGTCCTGGACTGGTTCACCGGGCGGCCGGGCATGTCCGGCGCCACCTTCTGCTTCGCCTCCTCCAGCGAG
GCGTACGCGGGAAGCGTCGCGGCCGGTCTCGCCGCGCTGCCCACCGGCGAGGACGTCCCGCTGCTGGTCCCCGAC
CCCGAGGTACCCCGCTCCTCGTACGGCTTCAGCAAGATCGCCGGAGAGCTGCTCTGCCGCACCTACGCCCAAGTC
CACGGCTTCGCCCTGCGCATGGTGCGCTTCCACAACGTCTACGGACCGCGCATGGGCTACGAACACGTCATCCCG
CAGTTCATCGAGCGCGTCCTCGGCGGCGCCGACCCCTTCGCCGTCTACGGCGGCGACCAGACCCGGGCCTTCTGC
CACGTCGACGACGCCGTGGCCGCCCTGATGGCGCTGGCCGCGCTGCCCACCAAGGAGACCCTGCTCGTCAACATC
GGCAACGACCAGGAGGAAGTCCGCATGGACGACCTCGCCCGGATGGTCTTCGAGACCGCCGGGCGCCGGCCGCGC
ATCGCCGCGCACCCGGCACCGCCCCTGTCCCCGGTGCGCCGCCTGCCCGACCTGACCCTGCTGCGCGAACTCACC
Appendix B
252
GGATACCGCCCCTCGGTCGATCTGCGCGAGGGGCTGCGCCGCACCTACGCGTGGTACGCGCACGACCTCGCCTCC
CGTGGAGCGGGGCGGTGA
> SPR_52980
GTGAGCGGCATGCGGGTGCGGTACGTGCACCAGGGGTACTTCCCCGCGCGAGCGGGCGCCGAGCTGATGACCCGG
TCCCTCGCCGTGGCCATGAGCCGCCGCGGGCTGCGCGTGGGCCTGTACGGCGGCGAGGGGGACCCGGAGGACGAG
CGGCTGATGAAGGCCGCGGGGATCGGCGTCGAACCGCTGCCGGTCCGGGACGGCGAGGAGCGTGCGGCGGACCTG
GTGCACGCCGTCGACGCCTTCCAGCCCGAGAACATCAGGACCGGCCTGCGTCTGGCCCGGGCCTGGGGCGTGCCG
TTCGCCGTGACACCGGCCTCCGCGCCGGACGTGTGGCCGCACCGCGCCGCCGTACTGGAGGGCTGCCGCCGCGCG
GACGCCGTGTTCGTCCTGACCGACGCGGAGCGCGACATGCTGCGCGCCGAAGGCGTGGCGGACTCCGTCCTGCAC
CGGATCGGCCAGGGAGCGCACCTGCCGGGCACCGCCGACCCGGAGGGGTTCCGTGCCGCGCACGGCATCAGCGGG
CCCGTGGTGCTCTTCCTCGGCCGCAAGATGCGCTCCAAGGGGTACCGGGTGCTGCTGGAGGCGACGCGGCACGTC
TGGGCCCGCCACCCCGAGGCGCACTTCGTCTTCCTCGGCCCGCGCTGGGACGAGGACTGGGCGCAGTGGTTCGCC
GCCCACGCCGACCCCCGGATCACCGAACTGGACCGGGTGGACGAGGACACCAAACTCAGTGCCCTGGCGGCCTGC
GACCTGCTCTGCCTGCCCTCGACGGTCGACGTCTTCCCCCTGGTGTTCGTCGAGGCGTGGATGTGCGGCAAGCCC
GTGATCGGCTCCGCGTTCATGGGCAGCGCGGAAGTGATCGCGGACGGGCGGGACGGGCTGATCGTCCCGCCCGCC
GCCCGGCCGGTGGCGGACGCGGTCAGCCGCCTCCTCGCCGACCCGGCCGAACGGGCGCGGATGGGGCGCGAGGGC
CATGACAGGGCGCGCCGCGAGCTCACCTGGGACGCGGTCGCCGCACAGGTGCACCGCGTCTACACCGAACTGGTC
CCGGCCCGGAGTTGA
> SPR_52990
GTGAAAGCAGTCGTACTGGCCGGCGGCGAGGGGCGGCGCCTGAGGCCCGCCACCTTCACCGTCCCCAAGCCCCTC
ATCGAGGTCGACGGCACCCCGATCCTGCACATCATCCTGCGACAGCTCAGAAGCGCCGGATTCACCCAGGTCACG
CTCTCGCTGGGCTATCGCGCCCAGCTGATCGAGGCCAGCTTCGACGGCCCCCGCTGGGCGGGGCTCGACCTGCGC
TTCTCCCTGGAGCACGAGCCGCTGGGGACCGCCGGGCCGCTGGGTCTGCTCGCGCCGCTGGAGGACTCCACCCTG
GTGATGAACGCGGACCTGCTCACCGACATCGACTTCGCCGATCTGTTCAGGCGGCACAAGAAGTCCGAGGCGGCG
GCGACGATCGCCCTCGTTCCGCGCCATGTCGACCTCGCCCACGGTGTGGTGGAGCTCGACGGCGAGAACCGGGTG
GCCGGCTTCCGGGAGAAACCGCGTATGAGCTTCCTGGCCAGCAGCGGCATCTACGTCCTGGAACCCTCGGTCCTG
CGTCTGCTGCCCCGGCGGGCGCGTTACGACATGCCGGCGCTGCTCAAGGACGCCTCCGCCCGGGGCGAGCGCGTC
GAGGGCCATGTCCTCGACGCCGCCTGGCACGACATCGGCACCCCCGAACAGCTCGCGGCGGCCGACGCCGCGCTC
CGTGCCGACCGGGCGCGCTATCTGGGCGCCCTGGAGCGGCACGGCCCCGGTGCCGAAACGGACTCCACGGAGGAG
GCGGTGCGCGGATGA
> SPR_53000
ATGAGCGACTGGAAGATCCCCCTGTACGGGCCGAGCACCGGTGCGGCGGAGGCCCGCGCCGTCGCCGAGGTCCTG
CGCGCCAACTGGCTGTCCGTGGGCCGGGTGACCCAGGACTTCGAGGAGCGCTACGCGGCCGCGCTGGACGTCGAG
GACGCGATCGCGGTCAGCAGCGGGACCGCGGCGCTGCACCTGGCCGTCCTCGCTCTGGGGATCGGCCCCGGCGAC
GAGGTCGTCCTGCCGTCACTGAGCTTCGTCTCGGCGGCCGCCGTGGTCGCCCTGTGCGGGGCCACACCGGTGTTC
GCCGAGGTGAGCGGCGCCCATGACCTGTGTGTGGACCCGGCGGACGTGGCCGCCCGGATCACGTCGCGCACCCGT
GCCGTCGTGGCGGTGCACTACGGCGGCCATACGGCCGACCTGCCCGCCCTGACCGAACTGGCCCGGCGGCACGGT
CTGGCCCTCATCGAGGACGCCGCGCACGCTCCGGTCACCAAGACCGCGCACGGCGTCCTGGGCACGGTCGGCGAC
ATCGGCTGCTACAGCTTCTTCGCCACCAAGAACCTGGCCATGGGGGAGGGCGGCGCCGTCGTCGCCCGCGATCCC
GCGGTGCGCGCCCGGATCAGGCGGCTGCGCTCGCACGCGCTGACCGTGGGCGCCGAGCAACGCCACCGCGGCGGG
CCCTCGGCGTACGACGTGGACGGGTTCGGCCTCAACTACCGCCCCACCGAGATCGCCTGCGCCCTCGGTCGCGTC
CAGTTGGAAGCGCTGGCGGAGCGCCGCATCCTGCGCCAGGAGGCGGTGCGGGCCTATCGCACGCTGCTCTCGGGG
CTGCCCGGCCTGGAAGTGCCCTTCGCGGAGCGCCCGGTGGAGGAGGGCGCGCACCATCTGTTCCCGCTCGTGCTG
CCCGACGGCCTCGACCGCGAAGACCTCCAGGCGCAGCTGCGGGCGGCCGGTGTGCAGAGCGGTGTGCACTACCCG
CCCACCCATCTGTTCACCGCCTACCGCGAACGTTTCGGCACCCGCCCCGGCAGCCTCCCGGTCACCGAGAGCGTC
GCCGCCCGGCAACTGTCGCTGCCGCTGCACGCCGGGACCGGGCTCCAGGACGTCGCCCATGTGGCCGAGGCGGTG
AGCCGGACATGGCCGCCGCAGCGGTGA
> SPR_53010
ATGGCCGCCGCAGCGGTGAGCCCCGCCTGTGGCACCCGTCTCGTCGAGGGGGTGCTGCACCGCGACGGACGTCCC
CTGTTCTGCGTCGGCGTCAACTACTTCCCCTCGCGGGCGGGCTGCGACTACTGGCGGGACTGGGACCCGGCCGTC
CTCGACGCGGACTTTGCCCGTATGGCCGCGCTCGGCTTCAACACCGTGCGGATCTTCGTGTTCTGGGCCGACTTC
GAGCCCACCGAGGGCAGCTACGACCCGCGTATGACCGCCAGGCTGCGTGAACTGGCGGTCCTGGCGGAACGACAC
CACCTGCTGGTTCTGCCCTCGCTGCTGACCATATGGATGAACGGGCAGCTCTTCGACCCGCCGTGGCGGGCGGGC
Appendix B
253
CGGGACCTGTGGCGCGATCCGGTCATGGCCGAGCGGCAGCGCGCCTTCGTCGGGCACATCGCGGGCACGCTGCGC
CACGCGCCCAACATCCTCGCCTACGACATCGGCGACGAGATCCCGCACGTCGACCCGGCCGCGTCCCACTCGCTC
GGCGCGCACGAGGTACGGGCATGGTGGGCCGACCTCGCCGAAGCGATCCGGACGGCGGACCCGGGGGCTTTGGTC
CTCCAGGCCAACGAAGGCTCGGCGGTCTTCGGCGACCACGCCTTCCGGCCCGAGCACGCCCGGCCGCTGGACCTG
GTCGCGCTGCACGGCTTCCCGCTGTGGACCCCCTTCCACATCGAATCGGCCGCCGCCGAGAAGGCCACCGCCTAT
CTGCCCTACCTGGTGCGGCGCGGGCGGGCCCACGCCCCGGTGCTCGTCGACGAGATGGGCAGCTACGGCTGCGAC
GAGGCCACCGCGGCCCGCTATCTGCGCGCCGCGGCCCACAGTGCCTTCGCCGCGGGCGCCGTCGGTATCTGCGTG
TGGTGCTGGCAGGACTTCACCTCCGAGAGCAAGCCGTACGCACTGCGGCCCGGCGAACGCTTCGTGGGGCTGCTG
GACATGGACGGCCGCGAGAAGCCCGCCATGGACGCCTTCCGCGGCTTCGCGCGCCGGGTGACCGGGGAACTCGCC
GGTTTCCGCCCGCTGCCGGCCCGGGTGGGCGTGTTCCTGCCGGAGCGGGCCCGCGACCACGACGGCGGGTACCTG
GCGTCCKGGGCCGACACGGACGCCGCCGCCTTCTACGCGCACTGTCTGCTCCAACAGGCCCATCTGCCCTACGAG
TTCACGGGGACCGAGGACCTGGAGCGGTACGCGATGGTGATCTGCCCCTCCGTACGGCCCCTGCCCCTGGCCGCG
CAGCGACGGCTCGGCGAGTACACGGTCGCGGGCGGGGTGCTGCTGTACTCCACCGGCGATCCGCTGGGCTCGGCG
GGCCTGGAGGAGCTCTTCGGTGTGCGGATCCGGGACTTCACCCTGAACACGGCCGAGCAGGACCGCTTCACCTGG
GCCGACACCCCTTTCCCGGTGCACTGGCCCGCCGGACGTATCCCCGTGGTCGACAGCACGCTCGCCGAGACGCTC
GCCCGCTACCCCAACGGCGCGCCCGCGCTGGCCCGCCGGCAGCGGGGCGGCGGCGTCGCCTACTTCCTCAACGCA
CCGCTGGAAGCCCTGCTGAACGCGCCCTACCGGCTCCAGGAGGCGCCCTGGCACCGGCTGTACGCCGCGATCGGT
GAGAAGGAAGGCATACGGCCGGAGCTGTTCGCCGACGAGCCGCTGGTGGAGACCACCGTCCTGGCGCGCGGCGAC
GAACGCCGCGGTGTGGTCGTCAACCACGCCACCGCGCCCGTGACGACGACGGTGTACCGCACGTCCGCCGCCGGA
GATCCACGGGCGGTGCGGCACCTGAGCCTGGAGCCCAAGGGCGTCGCGCTCGTGCGCTGGCACGACGGACCGCCC
CCACACCCCGCCGCCACGGGCTCCACAGCCGGGCAGGACGGCGAGCAATGA
> SPR_53020
ATGATCATCGACGCCCATGTCCACGCCGGTGAGTACTACCGCCACTTCACCGCCCGCTTCGCCGACCAGATGATG
GCCACCACCGGTCTGCCCCCCGAGGCGCTGTCCGCGCCGGAGGACAAGCTCCTGGCGGAGATGGACGCCGCCGGG
GTGGACCACGCCTTCCTGCTCGCCTTCGAGGTGCGGCGCGTCGAGGGGTTCTCGGTGCCGAACACCTTCGTCGCC
GAGCTGTGCGCCCGCCATCCCCAGCGGTTCACCGGCTTCGCCTCGGTGGACGCGGGCCGGCCCGGCGCCGCCGAG
GAACTACGCCACTCCGTGACCGAACTGGGCCTGCGCGGGCTGAAGACGGCCCCCTGCTACCTGCGGATGTCCCCG
GCCGACCCCCGCTGGTTCGAGGTGTACCGGACCGCCCAGGACCTGGGCATCCCGGTCCTCGTCCACACCGGCTAC
ACCCCCGCCAAGAACGCCGACGCCCGCTTCTTCTCCCCCTTGCTGCTGGAGCCGGTAGCGAAGCGGTTCCCGGAA
CTGCGGCTGATCCTGGCGCACTTGGGCACCCCGTGGACGGCCCAGTGCATCGATCTGCTCGCCCGCCATCCCCAT
CTGTACGCGGACCTGTCGATCTTCGGCTCCTACCAGTCGCCCCCCACGGTGGCCGCGGCGCTCGCCCACGCCCGC
GAACGGGGAGTGCTGGACCGCCTCCTGTGGGGCACCGACTTCCCCTTCGCCACCATGTCCGCCGCCGTGGCCCGG
ATGACCCGTCTCACCACCGACGCCGCGCCGTGGCCGTCGGACAGCGCCCCGCTGACACCCGACGAACACCGGGCG
GTCATGGGCGGCACCGCCGCACTACTGACCCAGAAATGA
> SPR_53030
GTGGCCTTCATCCAGGGATACGAATTCGACGCCATGTCGCGCCGCCCCCTGTACGGGGACGTGACCCAGCGGCTC
GTCGACCTGTGCGCGTGCGCCCCCGGCAGCGTCGTCGCGGACGTCGGCTGCGGATCGGGCCTGGCCACCGAGCTG
CTGCTGGAACGGTTCCCGCAGCTCGGCTCCGTGGTGGGCATCGACCCCTCCGACCACGAACTGGCCCTGGCCAAG
GAGCGGCTGCGGCACCACCCCCGGGCCCGCCTGGTCACCGGCCGGGCCCAGGACGTGGGCGCGATCGTCGGGCCG
GTGGACGCCGTGGTGCTCAGCAACGTCATGCACCAGATCCCCAGGGCCGAGCGCCCGGCAGTGCTGCGCGGCTGC
CACGATCTGCTCGGACCCGGCGGCCGGCTCGCTCTGAACACGCTGTTCTACGAGGGCGCCGTGCTGCCCGAGAGC
CGCCGCTTCTACGCCGCGTGGCTGCACGCCACCCGGACGTGGCTGCGCAGCCGGGGCGGCGACCTGGTGCTGGAG
CGGGAGGCCCCCATCGCCCTTGAGACCCTGACCCCGGCCGACCACGAAGCCCTCATGGCCGAGGCGGGCTTCGCC
TCCGTACGGTCCGAGGAGGACGTCTACCCGTGGACCCTGGAGGACTGGCAGGCCCTGTGCGGATACTCGGTCTTC
GTCGAGGGCGCCACCGGACTGACGGATGTGGACCTCGGCTCCCGCGCGCTGACGGCCGCCCTGCACACCGTCTTC
CGCGAGCTGGGGCTCAGCAGCGTGCCCCGGCGCTGGCTGTTCGTCCACGGCACGAGGCAGGGGTGA
> SPR_53040
GTGATCGCGGCGACGCGGCCCGCGGTGGGCACCGCCACCGGGCCGCTGTCCCACGCCCAGCTGGGCATGTGGTTC
CTGGAGCGGCGCGCGGGAACCACCTTGTACGCGGAACCGATGACCTTCCGCCTGGTCGGCGAGGTGAACGTGCGC
GCCCTGCACGCCGCGGTCGAGGAGGTCGTGCGCCGCCACGACGCGCTGCGCACCCGTTTCACCGTGGTCGACGGA
GCTCCCCGCCAGCACGTCGTCCCCGACGCGACGGTACCGATGGCCCGGATCGACCTGGCGTATCTGCCCCCCGCC
CGGCGCCACCAGCGCGCCGAGCGGCTCCTGCGCGCCGAGCTCGGCCGGCCCATCGACCTCGCCCAGGGACCGCTG
GCGCGGGCCACCCTGATCCGGCTCGCCCCGCACGAACACGTCTTCCGCCTCACCCTGCACCATCTCGTCTGCGAC
GCCTGGTCGTGGTGGATGGTCGTCCTGCGCGAGCTGGAGGAGGGCTACACCCGCCGGGTCCGCGGCGGGCACGCG
CCCCTGGAGCCCGTCCGCACCCACTACGCGGACTTCGTCGACTGGCAGGGACGGTGGCTTCAGAGCCCCGCGCAC
Appendix B
254
ACCCGGCAGGTCGCCCACTGGCGCCAGGCCCTCGCCGGTGTCGCCCCACTGCCCGAGCTCACCCTGGCCCGTACG
ACCGCGCCGGTGCCGTACACCTCCTCCGCGACCGAGTGGACGGCCTTCCCGCAGCCGCTGCACGACCGGCTGCGT
GCCCTCGGCCGCCGCGAACACGTCACCCTGTACATGCTGCTGCTCACCGCCTTCACGCGGGTGCTGCGCCGCCAT
GTGCCGGTGGACGACATCCTGGTCGGCACCCGGGGCGGGTTCCGCAGCCGGCCCGAGTTCGAGAAGACCGTCGGC
CTGTTCGTCAACATGCTGCCCATCCGCACACGTGTCACCGAGGGCACCGGGTTCCGCGAGCTGCTGCCGCGGGTG
CGCGACACCCTCCTGGGCACGTATCTGAACCGGGACCTGCCCTTCGAACGCCTCGTGGCCGAGCTGGGCCTGCGC
CGGACCGGACCCCGGCCGGTGATCAACGTGTGCGTCTCCTTCCAGACCACCCCCGAGGTCATGCCGCGACTCCCG
GGCCTGGACGTCACCTTGCTCAACCACGACCCCTACTCGCCGTTCGACCTCGATCTCGGCTTCTACACCGAGGAC
GGCGGGCTGCGCGCCCTGATGATCTACAACCCGGCCCGGTACACCCGCGACGCGGTGCGGCACCTGCTCGACGCG
CTGCACCGGGAGCTGTGGGACGCCACCCGCGCACCCGGGCCCGTCCGCGCCGACGACGAGAAGGAGACCACCGTA
TGA
> SPR_53050
ATGAGGGCTTTGCAGTGGCACGGCGCCCACCGGGTGGCCCTGACCGACGACGCCCCGCCGCCGCGCGTCGAGACA
CCGGACGACGCGGTCGTACGGGTGGTCAGAAGCGCGATCTGCGGCACCGACCTGCACCCCTACCGGGGCGAGATA
GCCTCCTTCACTCCGGGCACCGTCACCGGCCACGAGTTCACCGGGGTCGTCGAGTCCGTCGGCTCCGGGGTGCGC
GGCCTGCGGCCGGGACAGCGGGTCGTGGCCTCGGACATCATCGCCTGTGGCCGCTGCTGGTGGTGCCGGCACGGC
GGGCACTACCAGTGCGAGCAGGTGGGACTGTTCGGCTACGACACGGTCGTGGGCGCGCGCCCGTTCGCGGGCGGC
CATGCCGAGCTGGTCCGTGTCCCGTTCGCCGATGTGGTCCTCTTCCCGATCCCCGACGACGTCGGCGACGAGCGG
GCGCTGCTCATCGGGGACGCGCTGGCCACCGGGTACGCCTGCGCCCTGGGCGGCGAGGTGCGCCCCGGCGACACC
GTCGCGGTGATCGGGTGCGGCGCGGTGGGTCTGCTGGCGGCCGAGGCCGCCCGGCTGCTGGGAGCCGCCCGGATC
CTGGCCGTCGACCCCGTCGAGGCCCGCCGCAAAGCGGCCGCCGAGCGCGGTGCGACGGCCCTGGCCCCCGGGGAC
GACCTCTCGGAGCGGGTGCGCGAGCTGACACGGGGACGCGGCGCCGACGCCGTCCTTGAGGCGGTCGGCACGGAC
GCCGCCCTGCTGAGCGCGCTGGAGATCGTACGGCCGCGCGGCGCGGTGTGCGCGGTCGGCGCCCACGCCTCCACG
GCGATGCCGCTGCCGACCATCCAGGCCTTCGCGAAGGAGGTGACCCTGCGGTTCGCCGTGGGCGACCCCATCGCC
ACGCGTGAGCCGCTGATGGAGCTCGTCCGCCAGGACCGCGTCGACCCCTCCTTCGTCATCACCCACCGCCTGCCG
CTCGCCGAAGCCCCCGAAGCCTTCCGGCTGTTCGACGCCGGCGAGGCCATCAAGGTGGTGCTCGTCCCGTGA
> SPR_53060
GTGACCACCCGGCTCCCCGCGTCACCGGCGCGCCCGCCCGCCCCGGATCCGCTCGACGGCGCCGTTCACCGTCTC
GTCGAGGAGCGGGCCGCGCGCACACCGGACGCCGACGCCGTGCTGTGCGGGACCGAACGCCTCAGCTATGCGCAG
TTGGACCTGCGGGCCGAGCGTCTCGCACGCCGCCTGCGCCGCCACGGAGTGGGCCCCGAAGTCCCGGTGGGTGTA
CATCTGACGCGCTCGCCCGAACTGGCGGTGGCCTGTCTGGCGGTGCTCAAGGCGGGCGGGGCCTGCCTCCCGCTG
GACCCCGCCCATCCGCCCGGCCGTCTGGCCGACGCCCTGCGCGACGCCGGTGCCCCGCTGGTCCTGAGCCGGCGC
GCCCTGGCGGCGGGCCTGGGCCCGCACACGGCAGCCGTCCTCTACACGGACTCGGAGGCTGACGACTCGTCAGCC
CACGAGCGTCCGGCGCCCGGCCCGGCGGCAGGATCGCCGTCACCGGGTGCCCCGGCAGCCGAGGACCGGCGAGCG
GAGGAGCGGCCGGGCGACGCGGGGGCACCCCCCGCCCCGCCTCTTGCACGCCGCCTCGCCTGGGTCGCGTACACC
TCGGGATCCACCGGCCGACCCAAGCCCGTCGGCCTCGAACACGGGCCGCTCGCCAACCTCGCCGTGCAGATCGGC
CGCCGTCTGGACCTGGGCCCCGACGACCGCGTGCTGCAGTTCGCCTCCATCGGCTTCTCCGTCGCGGCCGAGGAG
ATGTTCTCGACCTGGGCCGCCGGAGCCTGTCTCGTCATCGATCCCGACGACACCCTCGCCGACAGCGCGGGGCTG
CTCACCGCCGTGGACAAGTACGCCGTCACCGTCCTCCAGCTCACCCCGTCCCTGTGGTACGAGTGGCTGCGCGAG
CTGAGCCGGGACGGCACTCTGCGCCCGCCCGCCTCGCTGCGGCTGCTCGTGGTGGGCAGCGAGCAGGCCGCACCC
GACCGGGCGGCCGACTGGCTGGCCACCGGGGTGCGCCTGGTGCACGAGTACGGTGCCACCGAGGGCACGGTCTCC
CAGCTGCTGTACGAACCCGACGTGTCACCGGCCGAGTTGAGGACCTGGCCGCGTCTGCCCGTCGGTACGCCGTTG
CCGGGTGTCCGCGTCCACATCCTCGACGCGCGGCGCCGACCGGTCCCCCCGGGCGAGCCCGGCGAGCTCCACCTC
GCCGGAGACACCCTGGCGCGCGGCTACCTGGGGCAGCCCGAGCTCACGGCCGAGCGCTTCCTGCCCGACCCGTTC
GCCGATCGGCCCGGCGCGCGCATGTACCGCACGGGCGACCTCGCACGACAGCGTGCGGACGGCACCATCGAGTTC
CTGGGTCGCGTCGACCACCAGATCACGCTGCGCGGCCTGCGTATCGAGCCGGGCGAGGTCGAGTCGGCGATCGGC
CGGTACCCCGGCGTCACCGAGAGCGCCGTGCTGGCCCGCACCACCGCACGGGGCGACGATCAGCTGTGCGCGTGC
GTGGTGTGGGAGAAGGAGCGCGACGAGGCCGGGCTGCGCGCCCACCTGCGCGCCGCGCTCCCGCCCGCGCTCGTC
CCCGCCCGCCTGCTGGCCCTGCCCGACCTGCCGCTGACCCCCCATGGCAAGGTGGACCGCCAGGCCCTGCGGGCC
CTGCGCTGGGAACCCGACCCCGTCGATTCACGGGGCGAGGCGCCGCGCACCGCGCTGGAGGCCGCCCTCGCCGCG
CTCTGGGCCTGGACCCTCGACGTGTCCCGGATCGGCCTCGACGACAGCCTCTTCGACCGGGGCGGCGACTCCCTG
ACCGCCACCCGGCTCGCGGCCCGACTGCGCGACGTCCTGATGGCGGACGTACGGCAGCGGACCGTGTTCGACGCG
CCGACCGTACGGGCCATGGCCGAGGTGATCTCGCGACAGCGCCGCCGCGCTCGCGCCGACGAGCCCGTGACGGCG
CCGGACGCGTACGAGGACGCCCCACTGACGTCGGCGCAGTGGCGCATGTGGCTGCACCACCGCAGATCCCCGACG
AGCGCCGCGTATCACGAACCCGTGGCCCTGCGGCTACGGGGCCCGCTCGACCCCGACCACCTCGTACGGGCACTG
CGGCGGACGGTGGGGCGGCATGCGGCTCTGCGCACCACCCTCGCCACGGCGGCGGGGCAGCCGCTCCAGCGGGTC
GCGCCGCCCGCCACCGCCGAGGCGTTCCCCCTGGCCCGCTTGGACCTGAGCGGCCACGCGGAGGGTGAACTTGAC
Appendix B
255
CGAGTGGTCGAGGAGTTGGCCGTGGCGCCGTTCGACCTGCGGCGTGGCCCCCTGATCCGCGCGCACCTGCTGCGG
GTCGCGGCCGACGAGCACGTCCTCGTGCTGGTCCTGCACCACATCGTCTTCGACGGATGGTCGATGGACGTGCTC
TTCCGGGATCTGGCGGCCTTCCACGACGAGGCCGCGACCGGAGTCGCCGCCGCGCCGGCGCCCCTGCCCTCCTCG
CCGCCCGCGCGCCCTCCGCTGCCCGCGGACGACCCGGGCGCCGAGCGCCGCGCCCAGGAACAGCGCGCGTACTGG
AAAAAGCAGCTGGCGGGGGCACCGCCCGTCACACGGCTGCCGACCGGCCGACCCACGTCCCGGCAGGGCACGGTG
CACTGCCCGTTCACCGTCCCGGCGGCCACCGCGGCGCGGCTGCGCGAGCTGGCGGCGGAGGAAGGCGCCACCCCG
TTCATGGTGATCCTCGCCGTCTTCACGGCAGTCGCCCACCAGAGCTCCGGCGCCCGCGACATCGTGGTGGGCACG
CTGCTGGCAGGCCGCGACGACCCGGACACCGCCGACCTGATCGGTCTGTTCACCCACACCGTTCCGCTGCGCACA
CAGGTGCCCGAGGGGACGACGGTCCGTCAGCTGGTGCGGCGGGTACGTGAGACGGTTCTGGGCGCGGTGGCCCAT
CAGGAGGTGCCGTTCGAGGACATCGTGGCGCACCTGGGACCGCCACGGGATCCCCGGCACAACCCGGTGTTCCAG
ATGGTGTTCTGCCACGGCGCCGCGTCCGCCCGCGCCCCGCAGCTGCGCGGCCTGAGCGTGACCCCCGTGGAGGTC
GCCGTGCCCTTCGCCAAGTTCGACGTCACCGTCATGGTCGACGAGGACCGGCACGGCGGATACGCCATCGACCTC
GCCTACGACCTCGCGCTCTACGACGAGACGACCGCTCACCGCCACACCGCGGCGTTCCGGTCCCTGCTGGAAGCC
GCCGCGGACCGACCGGAAGCAGGGGTGTGA
> SPR_53070
GTGTCCACAGAATCGACCGCCCGCACCACCGAATCCCCGGCCGCCGCCGCCCCGACGAACGCCGACGCACCCCCG
ACGCCCGAGGCCGCGTCGGTCCGGGTGGCCGAATCGGTTCGCCGGATCTGGGCCGAACTCCTGCAGATCGACGTC
GAAGCGATCGACGTCCGCCACAGCGACTTCTTCGAGCTCGGTGGCTATTCACTGCTCGCGCTCCAGGCCATCGGC
AGGCTGCTGGAGGAGCGGGGCTTCGACGAGTTCGAGGCGGCGGAGCTGGAGGGCGCGTTGCTCAACCGCCTCTTC
GAGGAGCCCACACCGTTGGCCCAGGCGGAGTGCCTGCAGTCCGCGCTGGCCGCCGGCGGGGCACCGCGCGCGTGA
> SPR_53080
ATGCACCACCGCTCCAAGGCCCTGTACGGGGTTTTCGTCACCGTCCTCGGACTCACGCTGACCGCCTGCGGCGGT
GACGGAGGAGGCGGGAAGTCCGGTGTTTCCGAGGGCGGGGCGGCGAAGGGCGAGACCGGCCAGGAAACCCCGTCC
GCGAGCCCGTCCGAGAAGCCGGAGTGGGGCCCCGCGCTGGCGATGGGCCAGCCGGCCCCGAAGCTGTACGAACCC
CTGAACACCAGCGGCGGGAAGTTCCAGGTAACCGCCAAGAAGATCGTCAAGGGAACTCCCCAGCAGGCAAAGGAC
CTGAACTGGGACACGCTCGCCCCGGGCGAACTCAAGGGCAGTACGCCGTACTTCGTGTACTTCACGTACACGCTC
AAGGAAGGCAAGCCGCAGGTCGCCAACCCTGACCTCGGGGCGCATGCCGCCGCCCTCGACATGAAGGGCGTGAAA
GTCGCGGAGCGGCCGGTCGTCCACTCGGGATTCGTCGACGGCGGTTGCGAAGTCCCACCCGTATACATGGGCTGG
GACATCGGCGAGACCCACACCCTCTGCACGGTCTTCGCCGGGGACGAGGCGCATCCCCCTGCCCGACTGGCCTGG
GGTACGGACATAGAAACCGACACGGACTACATGAAGGCCGCATCGTGGAAGTGGACCACCCAGTGA
> SPR_53090
ATGCCCGGTATGCGTGCGTTCTTCACCGTTGATCAGCGGCGGTTGTCGTTCGTGGACTTCGGCGGCCCGGGGCTC
CCGTTGCTGGCGTTTCACGGGCATTACAACGAGGCATCGGTGTTCGCCCCGCTGGCTGAAGCGCTGGCGCCGCAG
TGGCGGGTGATTGCCCTGGACCAGCGTGGGCATGGGGAATCCGACCGCGCCCCGAGCTACCAGCGGTCCGAGTAC
GGCGCTGACATCGCCGCCTTCCACCGCCATCTGGGACTCGGTCCGGTGGCGGTACTCGGGCATTCCCTGGGCGGC
GTGAACGCCTATCAGTACGCCGTCCGGCACGCGGACCGGGTGACAGCCCTGATCGTCGAGGACGTCGGCGCCGTG
GTGGATGCCGACTGGTCGCTCACCACGCAACTGCCTCGCAGAGCACCCTCCCGTCGGGCACTGGCCTCGGCTCTC
GGCCCGCTGGCGCCCTATCTCGAATGCACCTTCCGTCAGTTCGGTGACGGCTGGGGATTCTCCTTCGACATCAAG
GACACGGTGCTGTCCCACGAAGCCCTCACCGGGGACTACTGGGGCGACTGGGAGGCGGTGCCCTGCCCGACCCTG
CTGATCCGGGGAAGGGACAGCGTCGTCCTGGCGTCGGACCACGCCCGGGAGATGATAGCCCGCCGGGCAGGTCAG
GCCGAGCTGGTCGAACTTCCGGCCGGACACGTCGTGCACTGCGACGCTCCCGACGAGTTCGCCGCCGCTGTGCGC
ACCTTCCTGTCCCAACTGCCCGAGAGGTGA
Appendix C
256
APPENDIX C
The nucleotide sequences of the DNA fragments used to make each of the knock-out constructs
within the genes they target are provided in this appendix. The black text represents the regions
of the target gene within the wild-type strain, while the blue and green text represents the
sequences of the target gene that were integrated into the genome of each mutant strain via
homologous recombination. The position of the pOJ260 sequence within the target gene of
each mutant strain is also provided.
> S. polyantibioticus ∆CIN
CCGCATCACCGGCCTGTACGTGGCCCCGCCGATCGTGCTCGCCCTCGCCAAGCACCCGGCGGTCG//CCCAGTAC
GACCTCTCCTCCGTGGAGTACGTCGTCAGCGCCGCCGCCCCGCTGGACGCCGGGCTCGCCGCCGCCTGCTCGGCC
CGCCTCAAGGTCCCGCCGGTGCGCCAGGCGTACGGCATGACCGAGCTCTCGCCCGGCACCCACGCCGTCCCCCTG
TCGGCACAGAACCCGCCGCCCGGCACCGTCGGCACACTCCTGCCCGGCACCGAGATGCGGCTGCTCTGCCTCGAC
GACCCCGGCCGCGACGCCGCCCCCGGCGAGCGGGGCGAGATCGCCATCCGGGGCCCGCAGGTCATGAAGGGCTAC
CTCGGCCGCCCCGAGGCCACCGCCGAGATGATCGACGCCGAGGGCTGGGTGCACACCGGCGACGTCGGCCACGTA
GACGCCGACGGCTGGCTGTTCGTCGTCGACCGCGTCAAGGAGCTCATCAAGTACAAGGGCTTCCAGGTCGCCCCC
GCCGAACTGGAGGCGCTGCTGCTCACCCATGACGCCGTCGCGGACGCGGCGGTGGTCGGGGTGTACGACGAGGAC
GGCACCGAGGTGCCGTGCGCCTATGTCGTACGGGCACCCGGCGCCCCGGACCTCACCGCCGAGGACGTCATGGCG
TACGTGGCCGAACGCGTCGCCCCGTACAAGAAGATCCGCCGCCGC//pOJ260//GCCCAGTACGACCTCTCCTC
CGTGGAGTACGTCGTCAGCGCCGCCGCCCCGCTGGACGCCGGGCTCGCCGCCGCCTGCTCGGCCCGCCTCAAGGT
CCCGCCGGTGCGCCAGGCGTACGGCATGACCGAGCTCTCGCCCGGCACCCACGCCGTCCCCCTGTCGGCACAGAA
CCCGCCGCCCGGCACCGTCGGCACACTCCTGCCCGGCACCGAGATGCGGCTGCTCTGCCTCGACGACCCCGGCCG
CGACGCCGCCCCCGGCGAGCGGGGCGAGATCGCCATCCGGGGCCCGCAGGTCATGAAGGGCTACCTCGGCCGCCC
CGAGGCCACCGCCGAGATGATCGACGCCGAGGGCTGGGTGCACACCGGCGACGTCGGCCACGTAGACGCCGACGG
CTGGCTGTTCGTCGTCGACCGCGTCAAGGAGCTCATCAAGTACAAGGGCTTCCAGGTCGCCCCCGCCGAACTGGA
GGCGCTGCTGCTCACCCATGACGCCGTCGCGGACGCGGCGGTGGTCGGGGTGTACGACGAGGACGGCACCGAGGT
GCCGTGCGCCTATGTCGTACGGGCACCCGGCGCCCCGGACCTCACCGCCGAGGACGTCATGGCGTACGTGGCCGA
ACGCGTCGCCCCGTACAAGAAGATCCGCCG//GGTGGAGTTCGTCGCCGGGGTGCCGCGCGCGGCCACCGGGAAG
> S. polyantibioticus ∆LAC
GGAGCACGGTCTGCGCGGCACCCCGTACGGGCACTTCGGCGACGGCTGTGTCCACGTCCGGATCG//ACTTCGAC
CTGCTGAGTACGGCGGGTGTGGCGCGCTTCCGCCGGTTCTCCGAGGAGCTGGCGGACCTGGTGGCCGCGCACGGC
GGCTCGCTCTCCGGCGAGCACGGTGACGGGCAGGCGCGGGCCGAGCTGCTGCCGAGGATGTACGGGGACGAACTG
GTCGGGCTGTTCGGGCGGTTCAAGGACGTGTGGGACCCGTCGGGCCTGCTCAACCCCGGGATGCTGGCCCGTCCG
GCCCGGCTGGACGAGAACCTGCGCTTCGCGGTGCTGCCGAAGGGGCCGGTGGAGGTGGAGTTCGGCTATCCGCAG
GACGGCGGGGACTTCTCGGCGGCGGTACGGCGGTGTGTCGGGGTGGCCAAGTGCCGTACTGTGTCCGGGACTTCG
GGTTCGTCGGTGATGTGCCCGTCCTTCCGGGCCACCGGCGAGGAGGCGCACTCGACGCGGGGCCGGGCGCGGCTG
CTGCACGAGATGCTGGCGGGCGAGGTCGTCACCGGCGGCTGGCGCTCGCCGGAGGTGCGTGACGCGCTCGATCTG
TGCCTGTCCTGCAAGGGCTGCCGCAGCGACTGCCCGGTCGGGGTGGACATGGCCACGTACAAGGCGGAGTTCCTG
CCGC//pOJ260//GACTTCGACCTGCTGAGTACGGCGGGTGTGGCGCGCTTCCGCCGGTTCTCCGAGGAGCTGG
CGGACCTGGTGGCCGCGCACGGCGGCTCGCTCTCCGGCGAGCACGGTGACGGGCAGGCGCGGGCCGAGCTGCTGC
CGAGGATGTACGGGGACGAACTGGTCGGGCTGTTCGGGCGGTTCAAGGACGTGTGGGACCCGTCGGGCCTGCTCA
ACCCCGGGATGCTGGCCCGTCCGGCCCGGCTGGACGAGAACCTGCGCTTCGCGGTGCTGCCGAAGGGGCCGGTGG
AGGTGGAGTTCGGCTATCCGCAGGACGGCGGGGACTTCTCGGCGGCGGTACGGCGGTGTGTCGGGGTGGCCAAGT
Appendix C
257
GCCGTACTGTGTCCGGGACTTCGGGTTCGTCGGTGATGTGCCCGTCCTTCCGGGCCACCGGCGAGGAGGCGCACT
CGACGCGGGGCCGGGCGCGGCTGCTGCACGAGATGCTGGCGGGCGAGGTCGTCACCGGCGGCTGGCGCTCGCCGG
AGGTGCGTGACGCGCTCGATCTGTGCCTGTCCTGCAAGGGCTGCCGCAGCGACTGCCCGGTCGGGGTGGACATGG
CCACGTACAAGGCGGAGTTCCTG//CACCACCACTACCGGGGCCGGCTGCGCCCCGCCTCCCACTACGCGATGGG
> S. polyantibioticus ∆THI
ATGCCCGG//TATGCGTGCGTTCTTCACCGTTGATCAGCGGCGGTTGTCGTTCGTGGACTTCGGCGGCCCGGGGC
TCCCGTTGCTGGCGTTTCACGGGCATTACAACGAGGCATCGGTGTTCGCCCCGCTGGCTGAAGCGCTGGCGCCGC
AGTGGCGGGTGATTGCCCTGGACCAGCGTGGGCATGGGGAATCCGACCGCGCCCCGAGCTACCAGCGGTCCGAGT
ACGGCGCTGACATCGCCGCCTTCCACCGCCATCTGGGACTCGGTCCGGTGGCGGTACTCGGGCATTCCCTGGGCG
GCGTGAACGCCTATCAGTACGCCGTCCGGCACGCGGACCGGGTGACAGCCCTGATCGTCGAGGACGTCGGCGCCG
TGGTGGATGCCGACTGGTCGCTCACCACGCAACTGCCTCGCAGAGCACCCTCCCGTCGGGCACTGGCCTCGGCTC
TCGGCCCGCTGGCGCCCTATCTCGAATGCACCTTCCGC//pOJ260//GTATGCGTGCGTTCTTCACCGTTGATC
AGCGGCGGTTGTCGTTCGTGGACTTCGGCGGCCCGGGGCTCCCGTTGCTGGCGTTTCACGGGCATTACAACGAGG
CATCGGTGTTCGCCCCGCTGGCTGAAGCGCTGGCGCCGCAGTGGCGGGTGATTGCCCTGGACCAGCGTGGGCATG
GGGAATCCGACCGCGCCCCGAGCTACCAGCGGTCCGAGTACGGCGCTGACATCGCCGCCTTCCACCGCCATCTGG
GACTCGGTCCGGTGGCGGTACTCGGGCATTCCCTGGGCGGCGTGAACGCCTATCAGTACGCCGTCCGGCACGCGG
ACCGGGTGACAGCCCTGATCGTCGAGGACGTCGGCGCCGTGGTGGATGCCGACTGGTCGCTCACCACGCAACTGC
CTCGCAGAGCACCCTCCCGTCGGGCACTGGCCTCGGCTCTCGGCCCGCTGGCGCCCTATCTCGAATGCACCTT//
CCGTCAGTTCGGTGACGGCTGGGGATTCTCCTTCGACATCAAGGACACGGTGCTGTCCCACGAAGCCCTCACC
> S. polyantibioticus ∆A99
GGAGCGGCCGGGCGACGCGGGGGCACCCCCCGCCCCGCCTCTTGCACGCCGCCTCGCCTGGGTCGC//GTACACC
TCGGGATCCACCGGCCGACCCAAGCCCGTCGGCCTCGAACACGGGCCGCTCGCCAACCTCGCCGTGCAGATCGGC
CGCCGTCTGGACCTGGGCCCCGACGACCGCGTGCTGCAGTTCGCCTCCATCGGCTTCTCCGTCGCGGCCGAGGAG
ATGTTCTCGACCTGGGCCGCCGGAGCCTGTCTCGTCATCGATCCCGACGACACCCTCGCCGACAGCGCGGGGCTG
CTCACCGCCGTGGACAAGTACGCCGTCACCGTCCTCCAGCTCACCCCGTCCCTGTGGTACGAGTGGCTGCGCGAG
CTGAGCCGGGACGGCACTCTGCGCCCGCCCGCCTCGCTGCGGCTGCTCGTGGTGGGCAGCGAGCAGGCCGCACCC
GACCGGGCGGCCGACTGGCTGGCCACCGGGGTGCGCCTGGTGCACGAGTACGGTGCCACCGAGGGCACGGTCTCC
CAGCTGCTGTACGAACCCGACGTGTCACCGGCCGAGTTGAGGACCTGGCCGCGTCTGCCCGTCGGTACGCCGTTG
CCGGGTGTCCGCGTCCACATCCTCGACGCGCGGCGCCGACCGGTCCCCCCGGGCGAGCCCGGCGAGCTCCACCTC
GCCGGAGACACCCTGGCGCGCGGCTACCTGGGGCAGCCCGAGCTCACGGCCGAGCGCTTCCTGCCCGACCCGTTC
GCCGATCGGCCCGGCGCGCGCATGTACCGCACGGGCGAG//pOJ260//TTAAGTACACCTCGGGATCCACCGGC
CGACCCAAGCCCGTCGGCCTCGAACACGGGCCGCTCGCCAACCTCGCCGTGCAGATCGGCCGCCGTCTGGACCTG
GGCCCCGACGACCGCGTGCTGCAGTTCGCCTCCATCGGCTTCTCCGTCGCGGCCGAGGAGATGTTCTCGACCTGG
GCCGCCGGAGCCTGTCTCGTCATCGATCCCGACGACACCCTCGCCGACAGCGCGGGGCTGCTCACCGCCGTGGAC
AAGTACGCCGTCACCGTCCTCCAGCTCACCCCGTCCCTGTGGTACGAGTGGCTGCGCGAGCTGAGCCGGGACGGC
ACTCTGCGCCCGCCCGCCTCGCTGCGGCTGCTCGTGGTGGGCAGCGAGCAGGCCGCACCCGACCGGGCGGCCGAC
TGGCTGGCCACCGGGGTGCGCCTGGTGCACGAGTACGGTGCCACCGAGGGCACGGTCTCCCAGCTGCTGTACGAA
CCCGACGTGTCACCGGCCGAGTTGAGGACCTGGCCGCGTCTGCCCGTCGGTACGCCGTTGCCGGGTGTCCGCGTC
CACATCCTCGACGCGCGGCGCCGACCGGTCCCCCCGGGCGAGCCCGGCGAGCTCCACCTCGCCGGAGACACCCTG
GCGCGCGGCTACCTGGGGCAGCCCGAGCTCACGGCCGAGCGCTTCCTGCCCGACCCGTTCGCCGATCGGCCCGGC
GCGCGCATGTACCGCACGGGCGA//CCTCGCACGACAGCGTGCGGACGGCACCATCGAGTTCCTGGGTCGCGTCG
> S. polyantibioticus ∆CYC
GCGCGCCCTGCACGCCGCGGTCGAGGAGGTCGTGCGCCGCCACGACGCGCTGCGCACCCGTTTCAC//CGTGGTC
GACGGAGCTCCCCGCCAGCACGTCGTCCCCGACGCGACGGTACCGATGGCCCGGATCGACCTGGCGTATCTGCCC
CCCGCCCGGCGCCACCAGCGCGCCGAGCGGCTCCTGCGCGCCGAGCTCGGCCGGCCCATCGACCTCGCCCAGGGA
CCGCTGGCGCGGGCCACCCTGATCCGGCTCGCCCCGCACGAACACGTCTTCCGCCTCACCCTGCACCATCTCGTC
TGCGACGCCTGGTCGTGGTGGATGGTCGTCCTGCGCGAGCTGGAGGAGGGCTACACCCGCCGGGTCCGCGGCGGG
CACGCGCCCCTGGAGCCCGTCCGCACCCACTACGCGGACTTCGTCGACTGGCAGGGACGGTGGCTTCAGAGCCCC
GCGCACACCCGGCAGGTCGCCCACTGGCGCCAGGCCCTCGCCGGTGTCGCCCCACTGCCCGAGCTCACCCTGGCC
CGTACGACCGCGCCGGTGCCGTACACCTCCTCCGCGACCGAGTGGACGGCCTTCCCGCAGCCGCTGCACGACCGG
CTGCGTGCCCTCGGCCGCCGCGAACACGTCACCCTGTACATGCTGCTGCTCACCGCCTTCACGCGGGTGCTGCGC
CGCCATGTGCCGGTGGACGACATCCTGGTCGGCACCCGGGGCGGGTTCCGCAGCCGGCCCGAGTTCGAGAAGACC
GTCGGCCTGTTCGTCAACATGCTGCCCATCCGCACACGTGTCACCGAGGGCACCGGGTTCCGCGAGCTGCTGCCG
CGGGTGCGCGACACCCTCCTGGGCACGTATCTGAACCGGGACCTGCCCTTCGAACGCCTCGTGGCCGAGCTGGGC
CTGCGCCGGACCGGACCCCGGCCGGTGATCAACGTGTGCGTCTCCTTCCAGACCACCCCCGAGGTCATGCCGCGA
Appendix C
258
CTCCCGGGCCTGGACGTCACCTTGCTCAACCACGACCCCTACTCGCCGTTCGACCTCGATCTCGGCTTCTACACC
G//pOJ260//TTAACGTGGTCGACGGAGCTCCCCGCCAGCACGTCGTCCCCGACGCGACGGTACCGATGGCCCG
GATCGACCTGGCGTATCTGCCCCCCGCCCGGCGCCACCAGCGCGCCGAGCGGCTCCTGCGCGCCGAGCTCGGCCG
GCCCATCGACCTCGCCCAGGGACCGCTGGCGCGGGCCACCCTGATCCGGCTCGCCCCGCACGAACACGTCTTCCG
CCTCACCCTGCACCATCTCGTCTGCGACGCCTGGTCGTGGTGGATGGTCGTCCTGCGCGAGCTGGAGGAGGGCTA
CACCCGCCGGGTCCGCGGCGGGCACGCGCCCCTGGAGCCCGTCCGCACCCACTACGCGGACTTCGTCGACTGGCA
GGGACGGTGGCTTCAGAGCCCCGCGCACACCCGGCAGGTCGCCCACTGGCGCCAGGCCCTCGCCGGTGTCGCCCC
ACTGCCCGAGCTCACCCTGGCCCGTACGACCGCGCCGGTGCCGTACACCTCCTCCGCGACCGAGTGGACGGCCTT
CCCGCAGCCGCTGCACGACCGGCTGCGTGCCCTCGGCCGCCGCGAACACGTCACCCTGTACATGCTGCTGCTCAC
CGCCTTCACGCGGGTGCTGCGCCGCCATGTGCCGGTGGACGACATCCTGGTCGGCACCCGGGGCGGGTTCCGCAG
CCGGCCCGAGTTCGAGAAGACCGTCGGCCTGTTCGTCAACATGCTGCCCATCCGCACACGTGTCACCGAGGGCAC
CGGGTTCCGCGAGCTGCTGCCGCGGGTGCGCGACACCCTCCTGGGCACGTATCTGAACCGGGACCTGCCCTTCGA
ACGCCTCGTGGCCGAGCTGGGCCTGCGCCGGACCGGACCCCGGCCGGTGATCAACGTGTGCGTCTCCTTCCAGAC
CACCCCCGAGGTCATGCCGCGACTCCCGGGCCTGGACGTCACCTTGCTCAACCACGACCCCTACTCGCCGTTCGA
CCTCGATCTCGGCTTCTACACCG//GAGGACGGCGGGCTGCGCGCCCTGATGATCTACAACCCGGCCCGGTACAC
> S. polyantibioticus ∆ACY
ATGCCCTCGACCACTGAATCCGCCGCCACG//CTGTGGGAGCTCATCGACCGGGCCGTACGCCTCTCCCCCGCGG
CCACCGCCGTACGCCAGGGAACGCGGGCCCTCACCTTCCGCGAACTCGCCGACCGCGTCGAGACCACAGCACGCC
GCCTGCGCACGCGCCTGCCCGCCGACGGCGGAGGAACCGTGGGCCTGCTCTTCGAGAACACCGTGGAGAGCACGG
TGGCCTTCCTGGGCGCGCTGTACGCGGGCGTACCCCTCACGCCCCTCGAACCCGACAGCACCGAGCCCCATCTCC
TGGGCGTGCACCGGGACTTGGGACCCCTGCACCTCGTCGGTCGGCAGGCGAGGCTCAGCACACTGGCCCCTCCCG
CTGCCACCTCACTCGCCGCCCGGTGGCAGGGCGGTGTCCTCATCGACGTGGACGAGCTCACCGCCCGCCCCGGCG
CGGCGGCCCCGTCGGCGCCGCTGCCCGCGCCGCCGCCCGACGCCCCCGCGCTCTACCAGTACACCTCCGGCTCGA
CCGGCGAGCCGAGGGCCGCCGTGCACTCACAACACGACCTGGTCCGCGGTGGCGAGATCTACGCCCGCACCTACG
GCATCACCCCGGCCGACCGGATTCTCGCGGCCGTACCCCTGCTGCACTCCTTCGGCATGGTCGCCGCGCTCGCCA
CCGCGCTGCACGCCCGCGCGGAGCTCGTCCTGCTCGGCCGGTTCGCACCGGCCGAAATGCTCCGGGCACTGCACC
AGCACGCCTGCACCATCGTGGTGGGCACCCCGCTCGCCTATGACCTGGCGGCCCGTTCGGCGGCCTCGCGCAGCG
AGACGTCCCGCCCGGGCGACACCGTACGGCTCTGTCTGTCCTCCGGGGCGGCCCTGCCCCCGGCCGTGGCGGACC
GCTTCGCCCAGCACTGCGGCCCGGCCGTCCAGCAGGTCTACGGAAGCACCGAGG//pOJ206//TTAACTGTGGG
AGCTCATCGACCGGGCCGTACGCCTCTCCCCCGCGGCCACCGCCGTACGCCAGGGAACGCGGGCCCTCACCTTCC
GCGAACTCGCCGACCGCGTCGAGACCACAGCACGCCGCCTGCGCACGCGCCTGCCCGCCGACGGCGGAGGAACCG
TGGGCCTGCTCTTCGAGAACACCGTGGAGAGCACGGTGGCCTTCCTGGGCGCGCTGTACGCGGGCGTACCCCTCA
CGCCCCTCGAACCCGACAGCACCGAGCCCCATCTCCTGGGCGTGCACCGGGACTTGGGACCCCTGCACCTCGTCG
GTCGGCAGGCGAGGCTCAGCACACTGGCCCCTCCCGCTGCCACCTCACTCGCCGCCCGGTGGCAGGGCGGTGTCC
TCATCGACGTGGACGAGCTCACCGCCCGCCCCGGCGCGGCGGCCCCGTCGGCGCCGCTGCCCGCGCCGCCGCCCG
ACGCCCCCGCGCTCTACCAGTACACCTCCGGCTCGACCGGCGAGCCGAGGGCCGCCGTGCACTCACAACACGACC
TGGTCCGCGGTGGCGAGATCTACGCCCGCACCTACGGCATCACCCCGGCCGACCGGATTCTCGCGGCCGTACCCC
TGCTGCACTCCTTCGGCATGGTCGCCGCGCTCGCCACCGCGCTGCACGCCCGCGCGGAGCTCGTCCTGCTCGGCC
GGTTCGCACCGGCCGAAATGCTCCGGGCACTGCACCAGCACGCCTGCACCATCGTGGTGGGCACCCCGCTCGCCT
ATGACCTGGCGGCCCGTTCGGCGGCCTCGCGCAGCGAGACGTCCCGCCCGGGCGACACCGTACGGCTCTGTCTGT
CCTCCGGGGCGGCCCTGCCCCCGGCCGTGGCGGACCGCTTCGCCCAGCACTGCGGCCCGGCCGTCCAGCAGGTCT
ACGGAAGCACCGAG//GCCGGTGTCGTCGCCGCGCAGCTCCCGCAGCCGGACGGCACGGCCGATGCCGGGGTGGG
> S. polyantibioticus ∆PAAK
GGCGGTATGACGGCCCGCCAGGTCCAGCTGATCCAGGACTTCCGGCCCGAGGTCATCATGGTGACT//CCGTCGT
ACATGCTGACCCTCCTCGACGAGTTCGAGCGGCAGGGCGTCGACCCGCGCGCGACCTCGCTGAAAGTCGGGATCT
TCGGAGCCGAGCCGTGGACGGAGGAGATGCGCCGCGAGATCGAGGAGCGCTTCGCCATCGACGCGGTCGACATAT
ACGGGCTGTCGGAGGTGATCGGGCCCGGGGTGGCGCAGGAGTGCGTGGAGACCAAGGACGGGCTGCACATCTGGG
AGGACCACTTCTACCCGGAGATCGTCGACCCGCTCACCGGCGAGGTGCTGCCCGAGGGCGAGCGCGGCGAGCTGG
TCTTCACCTCGCTCACCAAGGAGGCCATGCCGGTGGTCCGCTACCGGACGCGGGACCTGACCCGGCTGCTGCCGG
GCTCGGCACGGGTGTTCCGGCGGATGGAGAAGGTGACCGGGCGCAGTGACGACCTGGTGATCCTGCGCGGGGTGA
ACCTGTTCCCCACCCAGATCGAGGAGATCGTGCTGCG//pOJ260//TTAACCGTCGTACATGCTGACCCTCCTC
GACGAGTTCGAGCGGCAGGGCGTCGACCCGCGCGCGACCTCGCTGAAAGTCGGGATCTTCGGAGCCGAGCCGTGG
ACGGAGGAGATGCGCCGCGAGATCGAGGAGCGCTTCGCCATCGACGCGGTCGACATATACGGGCTGTCGGAGGTG
ATCGGGCCCGGGGTGGCGCAGGAGTGCGTGGAGACCAAGGACGGGCTGCACATCTGGGAGGACCACTTCTACCCG
GAGATCGTCGACCCGCTCACCGGCGAGGTGCTGCCCGAGGGCGAGCGCGGCGAGCTGGTCTTCACCTCGCTCACC
AAGGAGGCCATGCCGGTGGTCCGCTACCGGACGCGGGACCTGACCCGGCTGCTGCCGGGCTCGGCACGGGTGTTC
CGGCGGATGGAGAAGGTGACCGGGCGCAGTGACGACCTGGTGATCCTGCGCGGGGTGAACCTGTTCCCCACCCAG
Appendix C
259
ATCGAGGAGATCGTGCTGC//GCACGCCCGGCCTCGCCCCCCACTTCCAGCTGCGGCTCACCAAGGAGGGCCGCC
TCGAC
> S. polyantibioticus ∆AD2
CCAGCCGACCGGGTGCTGGCTCAATGCCGGACCGAGCTGCCCGTCTACATCATTTATACCTCCGG//CTCCACCG
GCCTGCCGAAGGGTGTGGCAGTTCCGCACAGTTCGTGCGACAACATGGTGGAGTGGCAACGGACGCATTCGGTTC
GGCCCGACCTGAGGACTGCCCAGTACGCGCCGCTGAACTTCGATGTGTGCTTCCAGGAGATTCTCGGCACCCTGT
GCGGCGGCGGCACGCTCGTCGTAGTACCCGAGCGGCTCAGACGCGATCCGATCTCCCTGCTCGACTGGCTCGTGG
TGAACCGTATCGAGCGACTGTTCCTCCCCTGCGTGGCCCTTCATATGCTCACGGTCGCTGCCACCGCCGTGCATT
CACTCGCCGGTCTGGTCCTGGCGGAGATCAATGCCGCCGGTGAGCAGCTGGTCTGCACGCCGGCCATCAGAGATT
TCTTCGCGTTGCTGCCCGGTTGCCGACTGAACAACCACTACGGGCAGAGCGAATCGGCGATGGTCACGGTCCACA
CGCTGACCGGCCCCAGCCGGGAGTGGCCCGCGCTGGCTCCCATCGGCCGGCCACTGCCGGGATGCGAGGTGCTGA
TCGACCCCCCAGACCTCGAAGAGCCGGATGTCGGGGAACTGTTGGTGGCCGGAGCGCCTCTGTCGGCGGGATACC
TCAATCAGCCCCAACTCAGCGCCGAGCGGTATGTCACCGTCGATTCAACTCCGCAGGGACACACTCGTGCCTTCC
GGACGGGTGACCTCG//pOJ260//TTAACTCCACCGGCCTGCCGAAGGGTGTGGCAGTTCCGCACAGTTCGTGC
GACAACATGGTGGAGTGGCAACGGACGCATTCGGTTCGGCCCGACCTGAGGACTGCCCAGTACGCGCCGCTGAAC
TTCGATGTGTGCTTCCAGGAGATTCTCGGCACCCTGTGCGGCGGCGGCACGCTCGTCGTAGTACCCGAGCGGCTC
AGACGCGATCCGATCTCCCTGCTCGACTGGCTCGTGGTGAACCGTATCGAGCGACTGTTCCTCCCCTGCGTGGCC
CTTCATATGCTCACGGTCGCTGCCACCGCCGTGCATTCACTCGCCGGTCTGGTCCTGGCGGAGATCAATGCCGCC
GGTGAGCAGCTGGTCTGCACGCCGGCCATCAGAGATTTCTTCGCGTTGCTGCCCGGTTGCCGACTGAACAACCAC
TACGGGCAGAGCGAATCGGCGATGGTCACGGTCCACACGCTGACCGGCCCCAGCCGGGAGTGGCCCGCGCTGGCT
CCCATCGGCCGGCCACTGCCGGGATGCGAGGTGCTGATCGACCCCCCAGACCTCGAAGAGCCGGATGTCGGGGAA
CTGTTGGTGGCCGGAGCGCCTCTGTCGGCGGGATACCTCAATCAGCCCCAACTCAGCGCCGAGCGGTATGTCACC
GTCGATTCAACTCCGCAGGGACACACTCGTGCCTTCCGGACGGGTGACCTC//GTCCGGGTCGACGGGGACGTGC
> S. polyantibioticus ∆A16
CTGCCCGTGGTGCGGGCGGCCGACGCGCCCGAGGCGGCGGTGGACCGCGTCGCGGACGCGCACACC//GCGTACG
TGATGTACACCTCGGGATCGACCGGCCGCCCCAAGGGCGTGGCCGTGACGCACCGCAACATCCTGGCGCTCGCCG
CCGACCCCCTGTGGGCCGACGGCAGCCACACCCGGGTCCTCGCGCACGCGCCGCACTCCTTCGACGCCTCCACCT
TCGAGGTGTGGGTGCCGCTCCTTTGCGGCGGCACGGTCGTGGTGGCCCCGCCGTCCGACTCGGCGGCGCACGCCC
TGGAGCGGACGGTGGCACAGCACCGCCCGACCAGCGCCTTCGTCACCGCGTCCTTGTTCAACTCCCTGGTCGCCG
AGGGCAGTCCCGCCCTCGCCGGGCTGCAACACGTCCTGGTCGGCGGCGAAGCCCCTTCGGCGGCGGCCGTGCGGC
AGTTCCTGGCCGCCTCTCCCGGCACGGCGCTGACCAACGCCTACGGGCCGACCGAGAACACCACCTTCACGACCT
GCCACCGCTACGAGCCGGGCGCCGACGGCAGCCCCACGATCGGCCGGCCGATGGCCAACACCCGGGCCTACGTCC
TGGACGAGCGGCTGCACCCCGTACCCACCGGCGTCGTCGGCGAGCTGTACATCGCGGGCGCGGGCCTGGCCCGCG
GCTACCTCCACAACCCTGGCCTCACCGCCGGGCGGTTCGTCGCCGACCCCTTCGGCGCGCCGGGCGAGCGGATG/
/pOJ260//TTAAGCGTACGTGATGTACACCTCGGGATCGACCGGCCGCCCCAAGGGCGTGGCCGTGACGCACCG
CAACATCCTGGCGCTCGCCGCCGACCCCCTGTGGGCCGACGGCAGCCACACCCGGGTCCTCGCGCACGCGCCGCA
CTCCTTCGACGCCTCCACCTTCGAGGTGTGGGTGCCGCTCCTTTGCGGCGGCACGGTCGTGGTGGCCCCGCCGTC
CGACTCGGCGGCGCACGCCCTGGAGCGGACGGTGGCACAGCACCGCCCGACCAGCGCCTTCGTCACCGCGTCCTT
GTTCAACTCCCTGGTCGCCGAGGGCAGTCCCGCCCTCGCCGGGCTGCAACACGTCCTGGTCGGCGGCGAAGCCCC
TTCGGCGGCGGCCGTGCGGCAGTTCCTGGCCGCCTCTCCCGGCACGGCGCTGACCAACGCCTACGGGCCGACCGA
GAACACCACCTTCACGACCTGCCACCGCTACGAGCCGGGCGCCGACGGCAGCCCCACGATCGGCCGGCCGATGGC
CAACACCCGGGCCTACGTCCTGGACGAGCGGCTGCACCCCGTACCCACCGGCGTCGTCGGCGAGCTGTACATCGC
GGGCGCGGGCCTGGCCCGCGGCTACCTCCACAACCCTGGCCTCACCGCCGGGCGGTTCGTCGCCGACCCCTTCGG
CGCGCCGGGCGAGCGGAT//TACCGCACCGGGGACCTCGCACGGTGGAACGCGGACGGCGACATCGTCTTCACAG
> S. polyantibioticus ∆A18
GCCTTACAAGCGCCCCACGACGTGACCGATGGTGAGCGGGCGAGCGGGCTCACCGCCGACCACCCG//GCGTACG
TCATCTACACCTCCGGCTCGACGGGCAAGCCCAAAGGCGTGGTGATGACCCACCGCGGGCTCGCCAGTCTGGCCG
CCGACCACATCGAGCGGTTCGGGATCGTCGAGGGCGACGGGGTGCTCCAGTTCGCCTCGTTCAACTTCGACTGCT
CGGTGGGCGACCTGGTGATGGCGCTGGCCTCGGGCTCGGCGCTCATCGTACGGCCGCAGGACTGTCTGTCCGGGC
ACCAGCTGGGCGAGTTGATCGAGCGGACGTCCGCCACCCATGTGACGATCCCGCCGCAGGTCCTCGCCGCGCTGC
CGCCGGCCGCGCACCCCACCCTGAAGTCGGTCGCCACCGCCGGTGACGTGCTCACCGCCGAGCTCGTGGCCCAAT
GGGCGCCGGGGCGGCGGATGTTCAACGCCTACGGCCCGACCGAGACCACCGTGGACTCGCTGGCCACCGAGGTCG
AGGCCGGTTCGGGTGCCCCGCCGATCGGGCGGCCCCTGGTGAACACCCGGGTGTATGTGCTCGACGACGACATGC
GGCCGCTGCCCGTCGGCGCCGAGGGCGAGCTGTTCATCGCGGGTGCGGGGCTCGCCCGCGGCTATCTGCGTCAAC
CCGGGCTCACCGCCGAGCGGTTCGTGCCCTGCCCGTTCGGGGAGCCGGGCGAGCGCATGG//pOJ260//TTAAG
Appendix C
260
CGTACGTCATCTACACCTCCGGCTCGACGGGCAAGCCCAAAGGCGTGGTGATGACCCACCGCGGGCTCGCCAGTC
TGGCCGCCGACCACATCGAGCGGTTCGGGATCGTCGAGGGCGACGGGGTGCTCCAGTTCGCCTCGTTCAACTTCG
ACTGCTCGGTGGGCGACCTGGTGATGGCGCTGGCCTCGGGCTCGGCGCTCATCGTACGGCCGCAGGACTGTCTGT
CCGGGCACCAGCTGGGCGAGTTGATCGAGCGGACGTCCGCCACCCATGTGACGATCCCGCCGCAGGTCCTCGCCG
CGCTGCCGCCGGCCGCGCACCCCACCCTGAAGTCGGTCGCCACCGCCGGTGACGTGCTCACCGCCGAGCTCGTGG
CCCAATGGGCGCCGGGGCGGCGGATGTTCAACGCCTACGGCCCGACCGAGACCACCGTGGACTCGCTGGCCACCG
AGGTCGAGGCCGGTTCGGGTGCCCCGCCGATCGGGCGGCCCCTGGTGAACACCCGGGTGTATGTGCTCGACGACG
ACATGCGGCCGCTGCCCGTCGGCGCCGAGGGCGAGCTGTTCATCGCGGGTGCGGGGCTCGCCCGCGGCTATCTGC
GTCAACCCGGGCTCACCGCCGAGCGGTTCGTGCCCTGCCCGTTCGGGGAGCCGGGCGAGCGCATG//TACCGCAC
> S. polyantibioticus ∆A28
CGGTGCGTACGACAGCGCCGAACCGGCCGCCGTCGCCGTCGACATCGACGACTGGGCGTACGGCGG//ACCTCGG
GTTCCACGGGCAAGCCCAAGGGCGTCGTGACCGAGTACGCCGGACTCACCAACATGCTGATCAACCACCAGCGCC
GGATCTTCGAGCCGGTGCTGGCGGAGCACGGCGACCGGGTGTTCCGGATCGCCCACACCGTGTCGTTCGCGTTCG
ACATGTCGTGGGAGGAGCTGCTGTGGCTCGCCGACGGCCACGAGGTGCACATCTGCGACGAGGAACTGCGCCGCG
ACGCGCCCGCCCTGGTCGAGTACTGCGGCGAGCACGGGATCGACGTCATCAACGTGGCCCCCACGTACACGCAGC
AGCTGGTGGCCGAGGGCCTGCTCGACAACCCGGCCCGGCGCCCCGCGCTGGTGCTGCTGGGCGGCGAGGCGGTCA
CCCCGACCCTGTGGCAGCGGCTCGCCGGCACCGAGGGAACGGTCGGCTACAACCTGTACGGACCCACCGAGTACA
CCATCAACACCCTGGGCGTCGGCACCTTCGAGTGCGAGGACCCGGTGGTGGGCGTGGCGATCGACAACACCGACG
TGTTCGTGCTGGACCCGTGGCTGCGGCCGCTCCCGGACGGAGTTCCGGGTGAGCTCCACGTCACGGGCGTCGGCA
TCGCCCGCGGCTATCTGGGCTAGCACGCCCAGACCGCGCACCGGTTCGTGGCGTCCCCGTTCGGCGAGCCCGGCG
AGCGCATGTACCGCACCGGTGACCTCG//POJ260//TTAAACCTCGGGTTCCACGGGCAAGCCCAAGGGCGTCG
TGACCGAGTACGCCGGACTCACCAACATGCTGATCAACCACCAGCGCCGGATCTTCGAGCCGGTGCTGGCGGAGC
ACGGCGACCGGGTGTTCCGGATCGCCCACACCGTGTCGTTCGCGTTCGACATGTCGTGGGAGGAGCTGCTGTGGC
TCGCCGACGGCCACGAGGTGCACATCTGCGACGAGGAACTGCGCCGCGACGCGCCCGCCCTGGTCGAGTACTGCG
GCGAGCACGGGATCGACGTCATCAACGTGGCCCCCACGTACACGCAGCAGCTGGTGGCCGAGGGCCTGCTCGACA
ACCCGGCCCGGCGCCCCGCGCTGGTGCTGCTGGGCGGCGAGGCGGTCACCCCGACCCTGTGGCAGCGGCTCGCCG
GCACCGAGGGAACGGTCGGCTACAACCTGTACGGACCCACCGAGTACACCATCAACACCCTGGGCGTCGGCACCT
TCGAGTGCGAGGACCCGGTGGTGGGCGTGGCGATCGACAACACCGACGTGTTCGTGCTGGACCCGTGGCTGCGGC
CGCTCCCGGACGGAGTTCCGGGTGAGCTCCACGTCACGGGCGTCGGCATCGCCCGCGGCTATCTGGGCTAGCACG
CCCAGACCGCGCACCGGTTCGTGGCGTCCCCGTTCGGCGAGCCCGGCGAGCGCATGTACCGCACCGGTGACCTC
//ACGAGCTGCG
> S. polyantibioticus ∆A7
GCGCCGCCACCGCGCGCAGCCTGGCGTACGTCATCTACACCTCCGGCTCCACCGGCCGCCCCAAGG//GCCTACG
TGATCTACACCTCGGGGTCCACCGGCACGCCCAAGGGCGTGATGGTCGAACACCGCAATGCCACCCGGCTGTTCA
CCGCCACCGAGCCGTGGTTCGGCTTCGGCCGCGACGACGTGTGGACGCTGTTCCACTCCTTCGCCTTCGACTTCT
CCGTGTGGGAGATCTGGGGCGCGCTGCTGCACGGCGGCCGTCTGGTGATCGTGCCGCAGGCCACCACCCGCAACC
CGAACGACTTCTACGCCCTGCTGTGCGCGGAGGGGGTGACCGTCCTCAACCAGACGCCCAGCGCCTTCCGGCAGC
TGATCGCGGCCCAGGGCGACAGCCCGGCCGCACACCGGCTGCGCACGGTCGTCTTCGGCGGCGAGGCCCTGGACG
TCGCCGCACTCAAGCCGTGGCTGCGCCGCGCGGCCAACAAGGGCACCCGGCTCGTCAACATGTACGGGATCACCG
AGACCACCGTGCATGTCACCTACCGGCCGCTGACCGAGGCCGACGCGGAGCTCGCGGTGAGCCCGATCGGCGAAC
GGATCCCCGACCTGCGCACCTACGTCCTGGACCGGCACGGGCGGCCCGCCCCCGTCGGCGCGGTCGGCGAGCTGT
ATGTCGGCGGTGACGGCGTGGCCCGCGGCTACCTCAACCGGCCCGAGCTCACCGCCGAGCGCTTCCTGGACGACC
CGTTCTGCCCCGAGCCCGATGAGCGGATGTACCGCACCGGCGACGTCG//POJ260//TTAAGCCTACGTGATCT
ACACCTCGGGGTCCACCGGCACGCCCAAGGGCGTGATGGTCGAACACCGCAATGCCACCCGGCTGTTCACCGCCA
CCGAGCCGTGGTTCGGCTTCGGCCGCGACGACGTGTGGACGCTGTTCCACTCCTTCGCCTTCGACTTCTCCGTGT
GGGAGATCTGGGGCGCGCTGCTGCACGGCGGCCGTCTGGTGATCGTGCCGCAGGCCACCACCCGCAACCCGAACG
ACTTCTACGCCCTGCTGTGCGCGGAGGGGGTGACCGTCCTCAACCAGACGCCCAGCGCCTTCCGGCAGCTGATCG
CGGCCCAGGGCGACAGCCCGGCCGCACACCGGCTGCGCACGGTCGTCTTCGGCGGCGAGGCCCTGGACGTCGCCG
CACTCAAGCCGTGGCTGCGCCGCGCGGCCAACAAGGGCACCCGGCTCGTCAACATGTACGGGATCACCGAGACCA
CCGTGCATGTCACCTACCGGCCGCTGACCGAGGCCGACGCGGAGCTCGCGGTGAGCCCGATCGGCGAACGGATCC
CCGACCTGCGCACCTACGTCCTGGACCGGCACGGGCGGCCCGCCCCCGTCGGCGCGGTCGGCGAGCTGTATGTCG
GCGGTGACGGCGTGGCCCGCGGCTACCTCAACCGGCCCGAGCTCACCGCCGAGCGCTTCCTGGACGACCCGTTCT
GCCCCGAGCCCGATGAGCGGATGTACCGCACCGGCGACGTC//GTACGGCGCCTCGCGGACGGCACCCTGGAATT